The authors would like to acknowledge the European genomic institute for diabetes (ANR-10- LABEX-0046 to FP) and Qatar metabolic institute - Hamad medical corporation, Qatar. The authors thank as well the&#160;Medical Research Center at Hamad Medical Corporation for&#160; article processing fees support.

This protocol used discarded whole blood not fit for use as per institution discard guidelines. IRB approval and informed consent are not applicable.
1. Cooled cell processor setup
NOTE: All procedures must be done in a clean room environment, and staff must respect clean-room garment procedures.
<ol>
	<li>Purchasing of equipment
	<ol>
		<li>Purchase a spare plexiglass lid. Purchase the cooling equipment system, cooler with mechanically refrigerated gas stream temperature controller, air dryer, pressure and flow regulator, and the warranty (optional). See Table of Materials for references. 
		NOTE: The total cost is $16,133 without shipping or customs fees.</li>
		<li>Order medical dry air, a compressed medical gas that complies with standard grade (European pharmacopoeia reference 1238) from the local medical gas supplier. Purchase 2 cylinders of 6.8 m3 (47 L: 23 cm x 153 cm; diameter x length).</li>
	</ol>
	</li>
	<li>Set-up of the equipment (Figure 1 and Figure 2)
	<ol>
		<li>Enter the equipment in the GMP cleanroom (disinfect before entering the cleanroom according to GMP procedures.</li>
		<li>For the medical grade compressed air, install a gas line instead of placing the gas cylinder directly in the cleanroom.</li>
	</ol>
	</li>
	<li>Modification of the cell processor for cooling
	<ol>
		<li>First, remove the plexiglass cover. Keep the original plexiglass cover in case there is a problem with the warranty. Drill a hole in the spare lid of the cell processor with a diameter to match and fit the outer diameter of the cooling device nozzle tip to prevent cracking the centrifuge cover (13.3 mm recommended).</li>
		<li>Fix brackets together with a circular piece of foam. This is done to prevent breakage and/or scratching the lid, to hold the cooler nozzle in place above the cell processor, to protect the lid from excessive bending due to the high pressure of injected air, to support the weight of the nozzle, and lastly to ensure the maximum safety to the operator to have free and safe access to all required cell processor functions during the experimental procedure.</li>
		<li>Connect the cooling system to the cell processor by attaching the cooling nozzle to the hole in the plexiglass. Secure it with brackets and foam cushions.</li>
		<li>Place the refrigeration housing up to 8 feet (2.44 m) away from the nozzle. House the temperature controller in a separate cabinet and place it within 6 feet (1.83 m) of the refrigeration housing.</li>
		<li>Set the cooler set point at -40 &#176;C for cooling the air stream on the cooler. The exit air temperature is continuously indicated on the digital indicator to a resolution of 0.1 &#176;C. Set the outlet temperature digitally to 0.1 &#176;C with the temperature controller.</li>
		<li>Control the pressure and the flow rate of the air supply on the pressure and flow regulator module (Figure 2) before feeding to the cooler. Set them to 55 psi and 4 SCFM flow rates, respectively, to reach the optimum conditions on an air stream cooled at -30 &#176;C &#177; 2 &#176;C.</li>
		<li>Verify that the medical gas supply is regulated at an out pressure of 5 bar and can last at least 90 min, as this is required for the compressed air to reach -30 &#176;C. 
		&#8203;NOTE: Medical dry air is compressed medical gas, and fully pressurized dry air complies with standard grade.</li>
		<li>Deliver 5 bar from a cooler nozzle connected to the cell processor lid through a hole drilled in the centrifuge cover. Inject the cooled medical air inside the centrifugation bowl of the cell processor (Figure 2). Select the position of the air injection in a way that the cooled air is directed perpendicularly to the aluminum rim of the centrifuge rotor.</li>
		<li>When preparing the cell processor, ensure the rotor is at room temperature (18 &#176;C). Set the automatic thermo-regulator of the cooling system to -30 &#176;C and direct cool air injections to the rotor. 
		NOTE: Different trial conditions were tested to reach an optimum temperature gradient of +4 &#176;C, which is necessary to overcome the thermo-resistance of the cell processor cell separator system and heating of the rotor during centrifugation. The programmed thermo-regulator will maintain the optimum target temperature inside the cell processing machine during the procedure, particularly during the centrifugation, at a clean room temperature of +18 &#176;C.
		<ol>
			<li>Set up the cell processor with the first pre-cooling phase to cool down the processor (without the cell processor kit) at +3 &#176;C for 30 to 45 min, and the second pre-cooling phase of running the cell processor should be with the kit precooled at +4 &#176;C at 134 x g, for 5 min.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Airborne particulate count
<ol>
	<li>Assess the compliance of the cooling device with the GMP cleanroom environment as per ISO 14644-1 standard to evaluate the impact of heat devices during the operation phase.</li>
	<li>Use particle count monitoring to provide evidence that the required level of cleanliness is achieved at critical control points at rest and in operation.
	<ol>
		<li>The minimum number of sample locations depends on the area measured. Calculate this using the equation 
		<img alt="Equation 1" src="/files/ftp_upload/65321/65321eq01.jpg" style="margin: auto;" /> 
		&#8203;where A is the area of the cleanroom or clean air-controlled space in m2, and NL is the number of sample locations rounded up to a whole number.</li>
		<li>Airborne sampling volume is the minimum volume at each location. Ensure that the air volume is large enough to count 20 particles of the largest particle size specified. Calculate the airborne sampling volume through the equation 
		V= 20/C x 1000 
		where V is the minimum single sample volume per location, expressed in L, and C is the class limit (number of particles/m3).</li>
		<li>For each location, depending on the area, use one or more samples. Ensure the least volume sampled at each location is 2 L and the minimum sample time is at least 1 min.</li>
	</ol>
	</li>
	<li>Before sampling, purge the particle counter device with a purging filter. Remove the filter and place it with the isokinetic probe for sampling.</li>
	<li>Place the particle counter instrument in the identified sampling location. Initiate sample collection from the main screen of the particle counter device.</li>
	<li>Once the sampling is over, then take the particle counter device to the next location. When all locations for sampling are finished, review the data, and print a report.</li>
	<li>When the average particle concentration for all sampling points in the area falls below the class limit stated in Table 1, consider the room passed for having the allowable particle concentration.</li>
</ol>
3. Gradient preparation and cell separation processing
<ol>
	<li>Prior to the experimentation, precool the light and heavy density gradients and the cell processor kits overnight at +4 &#176;C in a refrigerator.</li>
	<li>Precool the cell processor for 45 min at rest (i.e., switch on the cell processor prior to use). Maintain the temperature between +2 &#176;C and +5 &#176;C. Insert the cell processor kit pre-cooled at +4 &#176;C.</li>
	<li>For the standard ethanol cooled double jacketed gradient maker (optional; Figure 3) connect the 2 glass chambers and place them on a magnetic stirrer. Clamp the tubing between the 2 chambers. Cool the gradient maker with an ethanol-circulating cooling chiller at 0 &#176;C.</li>
	<li>First, prepare to load the heavy layer at the bottom of the bag by filling the chamber with 130 mL of heavy density gradient (HD;1.087 g/mL). Load at a pump speed of 150 mL/ min.</li>
	<li>Once the heavy gradient is in the bag, add 130 mL of high density (HD) to the front beaker and 140 mL of low density (LD; 1.063 g/mL) gradients to the back (clamp closed).</li>
	<li>De-clamp the hemostat/clamp between the 2 chambers and create a continuous gradient at 50 mL/min pump speed.</li>
	<li>Prepare in advance human mononuclear buffy coat cells for purification from multiple pooled anonymous buffy coats collected from whole blood waste products (40 mL to 60 mL of whole blood was added to 10 buffy coat bags) depending on the availability from the blood bank.
	<ol>
		<li>MNC Separation
		<ol>
			<li>Use density gradient centrifugation for low-density human mononuclear cell isolation. To do this, follow the steps described below.</li>
			<li>For cell preparation, calculate the volume of blood (anticoagulated whole blood or suspended buffy coat). Dilute the blood with 2x-4x buffer solution by mixing inversion. The more diluted the blood, the better the purity of the mononuclear cells.</li>
			<li>Prepare buffer by mixing phosphate buffer with an anticoagulant. Calculate the number of tubes needed by dividing the volume of blood by 35 mL and pour 15 mL of 1.073 density gradient solution in each tube.</li>
			<li>Gradually layer 35 mL of the diluted blood suspension over the density gradient by gently running on the surface of the density gradient by using a 60 mL syringe connected to a needle; for small volumes, use a pipette. Try not to disrupt the interface, as this will result in a poor mononuclear layer.</li>
			<li>Centrifuge at 400 x g for 20 min at 20 &#176;C without any breaks (acceleration&#160;0, deceleration&#160;0). After centrifugation, gently harvest the interface layer of mononuclear cells using a sterile transfer pipette. Transfer the cells into another new 50 mL conical tube.</li>
			<li>Wash the cells 2x with buffer (PBS with added anticoagulant). For the first wash, fill the tube containing the cells with buffer till 50 mL. Centrifuge at 300 x g for 10 min at 20 &#176;C to pellet cells (acceleration&#160;9, deceleration&#160;0). Gently remove all supernatant carefully.</li>
			<li>Perform the second wash for the removal of platelets. Resuspend the pellet to a volume of 50 mL with buffer. Centrifuge at 200-300 x g for 10 min at 20 &#176;C (acceleration&#160;9, decceleration&#160;0).</li>
			<li>Reconstitute the cells to a final volume of 5 mL, these cells should have specific gravity resembling low-density MNC &#60;1.073. Measure the specific density of the gradient with a densiometer.</li>
			<li>Resuspend the pellet to a final volume of 100 mL with 95 mL of high-density gradient medium (1.083 g/mL) in a transfer bag. Use this product for purification using a cell separator. The final volume of 100 mL contains cells and 1.083 high-density gradient medium. 
			NOTE: The buffy coat may be stored in the refrigerator overnight in PBS containing 0.5% BSA or autologous plasma. The buffy coat can also be washed.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Top load the preformed gradients with a maximum of 30 mL of MNC isolated from the buffy coat suspended in 100 mL of concentrated conservation medium using a peristaltic pump at 25 mL/min. Keep the gradient at +4 &#176;C.</li>
	<li>Continuously measure the temperature inside the cell processor with a pre-calibrated thermoprobe. Using a digital thermocouple connected to a sterile thermoprobe, monitor the temperature of gradients and cells during the process.</li>
</ol>
4. Optimization of different steps of gradient loading
<ol>
	<li>Insert the cell processor kit pre-cooled at +4 &#176;C. Load the heavy gradient (130 mL) at + 4.5 &#176;C. At the end of gradient loading, set the centrifuge at a relatively low speed (537 x g) in order to minimize the heat generated by the rotor.</li>
	<li>Turn off the cell processor to allow the hydraulic system to go back to pre-run levels and release air.</li>
	<li>Restart the cell processor at 537 x g and add the heavy (130 mL) and low (140 mL) density gradients to create a continuous density gradient at +4.3 &#176;C and a flow rate of 50 mL/min.</li>
	<li>Load the buffy coat (30 mL) with 100 mL of cold storage medium at +6 &#176;C and a flow rate of 25 mL/min. Add 50 mL (25 mL x 2) of washing solution.</li>
	<li>After 5 min at +5 &#176;C, collect spun gradient in 12 bottles at a flow rate of 100 mL/min.</li>
	<li>Fill tubes with wash media (M199 + 20% human albumin + 10% Penicillin streptomycin). Fill tube 1 with 100 mL of wash media and 150 mL of cells and tubes 2-12 with 225 mL of wash media and 25 mL of cells.</li>
</ol>

Advancements in Cooling Cell Processors for Enhanced Viability in Cell Therapy

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	<li>Shapiro, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> The New England Journal of Medicine. 343 (4), 230-238 (2000).</li><li>Vesilind, G. W., Simpson, M. B., Colman, R. E., Kao, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+centrifugal+blood+cell+processor+for+washing+platelet+concentrates.">Evaluation of a centrifugal blood cell processor for washing platelet concentrates.</a> Transfusion. 28 (1), 46-51 (1988).</li><li>Lake, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+purification+of+human+islets+utilizing+discontinuous+albumin+gradient+on+IBM+2991+cell+separator.">Large-scale purification of human islets utilizing discontinuous albumin gradient on IBM 2991 cell separator.</a> Diabetes. 38, 143-145 (1989).</li><li>Ricordi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+islet+isolation+and+allotransplantation+in+22+consecutive+cases.">Human islet isolation and allotransplantation in 22 consecutive cases.</a> Transplantation. 53 (2), 407-414 (1992).</li><li>Friberg, A. S., Stahle, M., Brandhorst, H., Korsgren, O., Brandhorst, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+islet+separation+utilizing+a+closed+automated+purification+system.">Human islet separation utilizing a closed automated purification system.</a> Cell transplantation. 17 (12), 1305-1313 (2008).</li><li>Bampton, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Australian+experience+with+total+pancreatectomy+with+islet+autotransplantation+to+treat+chronic+pancreatitis.">Australian experience with total pancreatectomy with islet autotransplantation to treat chronic pancreatitis.</a> ANZ Journal of Surgery. 91 (12), 2663-2668 (2021).</li><li>Doppenberg, J. B., Engelse, M., de Koning, E. J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRISM:+A+Novel+Human+Islet+Isolation+Technique.">PRISM: A Novel Human Islet Isolation Technique.</a> Transplantation. 106 (6), 1271-1278 (2021).</li><li>Shimoda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+purification+method+using+large+bottles+effectively+achieves+high+islet+yield+from+pig+pancreas.">Islet purification method using large bottles effectively achieves high islet yield from pig pancreas.</a> Cell Transplantation. 21 (2-3), 501-508 (2012).</li><li>Noguchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+Islet+Purification+from+Large+Mammals+and+Humans+Using+a+COBE+2991+Cell+Processor+versus+Large+Plastic+Bottles.">Pancreatic Islet Purification from Large Mammals and Humans Using a COBE 2991 Cell Processor versus Large Plastic Bottles.</a> Journal of Clinical Medicine. 10 (1), 10 (2021).</li><li>Shapiro, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+trial+of+the+Edmonton+protocol+for+islet+transplantation.">International trial of the Edmonton protocol for islet transplantation.</a> The New England Journal of Medicine. 355 (13), 1318-1330 (2006).</li><li>Ricordi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+Institutes+of+Health-Sponsored+Clinical+Islet+Transplantation+Consortium+Phase+3+Trial:+Manufacture+of+a+Complex+Cellular+Product+at+Eight+Processing+Facilities.">National Institutes of Health-Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities.</a> Diabetes. 65 (11), 3418-3428 (2016).</li><li>Swift, S., Kin, T., Mirbolooki, M., Wilson, R., Lakey, J. R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+cooling+systems+during+islet+purification.">Comparison of cooling systems during islet purification.</a> Cell Transplantation. 15 (2), 175-180 (2006).</li><li>Rilo, H. L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+islet+number+and+viability+obtained+during+purification+on+Euro-Collins-Ficoll+gradients+processed+on+a+refrigerated+or+nonrefrigerated+COBE+2991+cell+separator.">Comparison of islet number and viability obtained during purification on Euro-Collins-Ficoll gradients processed on a refrigerated or nonrefrigerated COBE 2991 cell separator.</a> Transplantation Proceedings. 26 (6), 3430 (1994).</li><li>Chadwick, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+islet+purification+using+bovine+serum+albumin:+The+importance+of+density+gradient+temperature+and+osmolality.">Pancreatic islet purification using bovine serum albumin: The importance of density gradient temperature and osmolality.</a> Cell Transplantation. 2 (4), 355 (1993).</li><li>Nano, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+human+pancreatic+islet+isolation+around+Europe:+results+of+a+survey+study.">Heterogeneity of human pancreatic islet isolation around Europe: results of a survey study.</a> Transplantation. 104 (1), 190-196 (2020).</li><li>Chadwick, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Storage+of+pancreatic+digest+before+islet+purification.">Storage of pancreatic digest before islet purification.</a> Transplantation. 58 (1), 99-104 (1994).</li><li>Amrani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detrimental+effects+of+temperature+on+the+efficacy+of+the+University+of+Wisconsin+solution+when+used+for+cardioplegia+at+moderate+hypothermia.+Comparison+with+the+St.+Thomas+Hospital+solution+at+4+degrees+C+and+20+degrees+C.">Detrimental effects of temperature on the efficacy of the University of Wisconsin solution when used for cardioplegia at moderate hypothermia. Comparison with the St. Thomas Hospital solution at 4 degrees C and 20 degrees C.</a> Circulation. 86, 280-288 (1992).</li><li>Fremes, S. E., Li, R. K., Weisel, R. D., Mickle, D. A., Tumiati, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+hypothermic+cardiac+storage+with+University+of+Wisconsin+solution.+An+assessment+with+human+cell+cultures.">Prolonged hypothermic cardiac storage with University of Wisconsin solution. An assessment with human cell cultures.</a> The Journal of Thoracic and Cardiovascular Surgery. 102 (5), 666-672 (1991).</li><li>Adewola, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+cooling+systems+for+the+COBE+2991+cell+separator+used+in+the+purification+of+human+pancreatic+islets+of+Langerhans.">Comparing cooling systems for the COBE 2991 cell separator used in the purification of human pancreatic islets of Langerhans.</a> Cryo Letters. 31 (4), 310-317 (2010).</li><li>Keymeulen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+standardized+beta-cell+grafts+in+a+liver+segment+of+IDDM+patients:+graft+and+recipients+characteristics+in+two+cases+of+insulin-independence+under+maintenance+immunosuppression+for+prior+kidney+graft.">Implantation of standardized beta-cell grafts in a liver segment of IDDM patients: graft and recipients characteristics in two cases of insulin-independence under maintenance immunosuppression for prior kidney graft.</a> Diabetologia. 41 (4), 452-459 (1998).</li><li>de Kort, H., de Koning, E. J., Rabelink, T. J., Bruijn, J. A., Bajema, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation+in+type+1+diabetes.">Islet transplantation in type 1 diabetes.</a> BMJ. 342, (2011).</li><li>Barbaro, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+human+pancreatic+islet+purification+with+the+refined+UIC-UB+density+gradient.">Improved human pancreatic islet purification with the refined UIC-UB density gradient.</a> Transplantation. 84 (9), 1200-1203 (2007).</li><li>Lemarie, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+single-platform+method+for+the+enumeration+of+CD34++cells.">A new single-platform method for the enumeration of CD34+ cells.</a> Cytotherapy. 11 (6), 804-806 (2009).</li></ol>

The authors declare no conflict of interest.

Islet isolation facilities&#160;around the world have adopted for decades human islet purification in a repurposed cell processor. Although some islet production facilities continue to process islets in an unrefrigerated cell processor, most centers use a refrigerated version of the cell processor10,11,15. Centers either place the cell processor in a cold room which has the major disadvantage of exposure to humidity and condensa...

Islet transplantation for severe Type 1 diabetes can restore endogenous insulin secretion and has become the standard of care in a number of countries1. Islet isolation is a multi-step procedure that includes warm enzymatic and mechanical digestion of the pancreas, followed by cooled phases for recovery of digested tissue and islet purification with continuous or discontinuous density gradients on a cell processor. Rapid cooling slows the enzymatic digestion phase and protects islets from digestive enzymes liberated by contaminating acinar cells. The cell processor was originally designed to separate cellular blood components at ambient temperature2. It was repurposed to purify human pancreatic islets of Langerhans in 19893. For three decades, human islets have been purified for clinical trials and, more recently, standard-of-care, either in a cell processor with discontinuous gradients4 or continuous density gradients1 made with a gradient maker1 or equivalent computer-operated mixer5,6,7. Other groups have reported islet purification from large mammals and humans using large cylindrical plastic bottles8,7,9, which substantially improved the efficacy of purification and islet quality.
Indeed, considering the gold standard to purify human islets for clinical transplantation10,11, the manufactured cell processor was without refrigeration. As a direct consequence of the lack of refrigeration, islets were exposed to increasing temperatures during the purification step. During centrifugation, an uncontrolled increase in temperature occurs due to the mechanical friction of the rotor12,13. Cell processors were adapted with a cooling system in islet isolation centers that statistically increased glucose-induced insulin secretion and cell viability, confirming the advantage of keeping pancreatic islets at low temperatures during the purification procedure14,15.
It should be noted that in the late 1980s, 10 refrigerated prototypes were built to reduce islet damage during density gradient purification but received no further attention. The cell processor prototypes were based on spindle and bowl cooling with chilled polyethylene glycol. In particular, islet purification solutions, including organ preservation solutions and polysaccharide density gradients, may be deleterious for islet viability when used at or above room temperature16,17,18. A comparison of different cooling strategies of the cell processor for islet cell purification versus the original non-refrigerated cell processor was evaluated to combat the uncontrolled increase of temperature that occurs due to the friction of the rotor. Several attempts have been made to cool down the cell processor during human islet purification. The cell processor can be temporarily or permanently housed in a specially designed cold room to maintain a temperature of 0-8 &#176;C19 during the gradient centrifugation which is currently used&#160;at the islet isolation centers at the University of Illinois Chicago19, University of Miami14, University of Oxford facilities15, and the University of San Francisco (the custom cooling unit encases the cell processor). Another modification includes core cooling of the cell processor spindle with a cooling system using electronically controlled liquid nitrogen cooling in Geneva, Sweden, and the Czech Republic15,19. Another alternative in the mid of 2000 was the core cooling of the cell processor with an air-conditioning unit12 or by liquid cooling of the shaft at the University of Lille.
Cooling modifications of the cell processor are not readily available and come with risks (humidity, condensation build-up) inside GMP cleanroom environments, including loss of warranty and refusal of the constructor to service the machine. Consequently, some islet centers, including Milan, Italy, Oslo, Norway, and Edinburgh, Scotland, do not use a cooling system for cell processors during the islet purification process15.
The current study was designed to test the effectiveness of a newly developed readily available cooler in conjunction with a minimally modified cell processor system. Briefly, the air-based cooler described here is a mechanically refrigerated heat exchanger controlling the temperature of 1 standard cubic foot per minute (SCFM) of dry gas between -40 &#176;C to -100 &#176;C. This cooling system requires a dry medical air supply. The air will flow through the line and heat exchanger and out through the (7.9 mm) nozzle. A heater located within the nozzle supplies heat to control the temperature of the air stream. A spare solid plastic centrifuge cover was purchased, and minimal modifications were made. A hole of 13.3 mm diameter was drilled in the cover, and cooled air was delivered from the cooler nozzle to the cell processor via the hole in the centrifuge cover. The cooled medical air was injected inside the centrifugation bowl of the cell processor with constant control of the temperature of the liquids or density gradients.
We evaluated the technical performance of the cooled cell processor and the impact of the cooling element on the GMP cleanroom environment. The main focus was to achieve a robustly low (&#60;10 &#176;C) temperature during human islet purification to preserve viability. Using a buffy coat density separation as a proxy for islet isolation, we determined the efficacy of this cooling protocol on cell viability.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Air particles counter</td><td>Lasair III&amp;nbsp;</td><td></td><td></td></tr><tr><td>AirJet XR40&amp;nbsp;</td><td>SP Scientific</td><td>FTS system XR40</td><td></td></tr><tr><td>Biosafety Cabinet</td><td>Thermo Scientific</td><td>EQ-1301</td><td></td></tr><tr><td>Circulating Cooling Chiller</td><td>Julabo</td><td>CF30</td><td></td></tr><tr><td>COBE 2991&amp;nbsp;</td><td>Terumo BCT</td><td></td><td></td></tr><tr><td>Conical Cooling Tray</td><td>Biorep</td><td>CCT-01</td><td></td></tr><tr><td>Double jacketed gradient maker&amp;nbsp;</td><td>customized in house</td><td></td><td></td></tr><tr><td>KJT-Thermocouple Thermometer</td><td>Hanna Instruments</td><td>HI93551N</td><td>For measuring liquids temperature</td></tr><tr><td>Magnetic stirrer&amp;nbsp;</td><td>Thermo Scientific</td><td>88880014</td><td></td></tr><tr><td>Peristaltic pump&amp;nbsp;</td><td>MasterFlex</td><td>MK-77921-79</td><td></td></tr><tr><td>Thermometer&amp;nbsp;</td><td>Extech Instruments</td><td>RMS 430</td><td>For COBE temperature</td></tr><tr><td>Waterless Bead Bath</td><td>Cole-Parmer</td><td>10122-00</td><td></td></tr><tr><td>&lt;strong&gt;Materials and reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BD stem cell enumeration kit</td><td>BD Biosciences&amp;nbsp;</td><td>344563</td><td></td></tr><tr><td>COBE 2911 tubing kit</td><td>Terumo BCT</td><td>90819</td><td></td></tr><tr><td>Conical Tubes 250ml</td><td>Corning</td><td>430776</td><td></td></tr><tr><td>Gradient density 1.1</td><td>Biochrom&amp;nbsp;</td><td>L6155</td><td></td></tr><tr><td>Graduated disposable bottles</td><td>Thermo fisher</td><td>382019-1000</td><td></td></tr><tr><td>Human albumin 20%&amp;nbsp;</td><td>Kedrion Biopharma&amp;nbsp;</td><td>8091600</td><td></td></tr><tr><td>M199 washing solution&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>99-784-CM&amp;nbsp;</td><td></td></tr><tr><td>Masterflex tubes</td><td>Masterflex</td><td>96400-6</td><td></td></tr><tr><td>Medical Dry Air</td><td>Linde Healthcare</td><td>Medi On 22</td><td></td></tr><tr><td>Pencillin-streptomycin&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>Thermoprobe</td><td>Biorep</td><td>TC-02</td><td>Thermosensor</td></tr><tr><td>Trypan blue&amp;nbsp;</td><td>Sigma Aldrich</td><td>15250061</td><td></td></tr><tr><td>University of Wisconsin Belzer (UW)&amp;nbsp;</td><td>Bridge of Life&amp;nbsp;</td><td>RM/N 4055</td><td>Conservation Medium</td></tr></tbody></table>

evaluation of the impact of a new cooling cell processor system on islet cell isolation facility

Standard cell therapy equipment, including the gold standard cell processor to purify human islets for clinical transplantation, is rarely refrigerated, potentially exposing cells to elevated temperatures during the centrifugation step. Custom cooling systems have a direct benefit on human islet viability and function. The current study was designed to test the effectiveness of a newly developed, readily available cooled cell processor system requiring minimal modifications and to evaluate its impact on human cell viability and the GMP cleanroom environment.&#160;The cooler system, a mechanically refrigerated heat exchanger set at -30 &#176;C was used to deliver cooled medical grade dry air to the cell processor bowl through a hole drilled in the centrifuge cover. With the limited availability of pancreas donors in Qatar, system validation was done with continuous density gradient purification of pooled human bone marrow buffy coat. Sterility, turbulence, and particle count were measured in class C and class B clean room environments. No turbulence developed around the cooled cell processor, and no excess 0.5 &#181;m and 5 &#181;m airborne particulates were generated as per cleanroom GMP standards. At the beginning and end of the collection steps, the temperature rose respectively to 21.50 &#176;C &#177; 0.34 &#176;C and 21.93 &#176;C &#177; 0.20 &#176;C in the non-cooled cell processor and to only 10.9 &#176;C &#177; 0.17 &#176;C and 11.16 &#176;C &#177; 0.35 &#176;C in the cooled- cell processor (p &#60;0.05). The cooled cell processor led to both improved recovery (98%) of the mononuclear cell fraction and viability (100% &#177; 2%) post-processing. The new cooling system effectively reduces the heat produced by the cell processor while having no particulate impact on the GMP clean room environment. The cooled cell processor described here is an inexpensive ($16,000 without including taxes, customs clearance, and transportation) and minimally invasive method to provide robust cooling. Currently, this technology in the GMP cell therapy facility&#160;is being applied to human islet cell isolation and transplantation for the clinical program.

The impact of the cooled cell processor on the GMP cleanroom was first quantified. Standardized methods used a particle counter measuring the airborne particle contamination in a GMP grade class C and class B clean room environment at critical points in proximity to the cell processor and pressurized air cooler system. Results demonstrated that no turbulence developed around the equipment. Recorded data showed that there was no excess of 0.5 &#181;m and 5 &#181;m airborne particulate as per cleanroom GMP grade C, grade B...

Watch this Scientific Journal Video about Evaluation of the Impact of a New Cooling Cell Processor System on Islet Cell Isolation Facility at JoVE.com

Evaluation of the Impact of a New Cooling Cell Processor System on Islet Cell Isolation Facility

The protocol here describes a custom cooling cell processor system that is compatible with a good manufacturing practices (GMP) cleanroom to improve cell viability post-processing.

Standard cell therapy equipment, including the gold standard cell processor to purify human islets for clinical transplantation, is rarely refrigerated, ...

evaluation-impact-new-cooling-cell-processor-system-on-islet-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

191 次观看.  Hamad Medical Corporation. The protocol here describes a custom cooling cell processor system that is compatible with a good manufacturing practices (GMP) cleanroom to improve cell viability post-processing.

视频: Evaluation of the Impact of a New Cooling Cell Processor System on Islet Cell Isolation Facility

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

OLE Isacson：帕金森氏病的新疗法的发展

科研

生物学

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intestine and the pancreas with a midline incision. The diaphragm will also be cut to verify that the mouse will be sacrificed.Before we get started, I'd like to point out some of the important structures. The stomach is lying right here with the pancreas attached underneath into the duodenum and above. This is the liver where we'll find the gallbladder and the common bile duct, which you can see right here.So what Pavo will do next is he will isolate the small intestine, find where the common bile duct enters the small intestine. Once the intestine is exposed, we clamp on either side of the common bile duct with mosquito clamps. We find that this allows more collagenates to enter the pancreas while other groups try to direct the clamp on top of the common bile duct.We find that this is a better technique in allowing enzyme to enter the pancreas. This is a gentle procedure. You don't want to tear the bowel.You wanna allow the clamps to lie gently on either side of the common bile duct. Now we will find a gallbladder, and once the gallbladder's exposed, we will use that as a guide to injecting the collagenase into the common bile duct. Powell's using a 30 gauge needle with a five cc syringe containing three mills of collagenase solution.As he's entering the common bile duct, he's distending that duct with collagenase solution and then he travels down the common bile duct till he reaches near the intestine. And as he's going down, the enzyme solutions being pumped into the injected into the pancreas. Now that the pancreas is inflated with collagenase solution, Powell's gonna remove the hemostats and then remove the pancreas from the the mouse carefully tearing it away from the intestine.He removes it from the stomach. Okay, and then using the spleen as a handle, he lifts up the pancreas and then slowly snips it away in the common bile duct, and that's it. Pancreas is laid out and then the spleen is removed, and there he's removing the meso enteric tissue, and then the pancreas is placed in a two mill collagenase solution and a sterilized glass vial and it's placed back on Ice.The isolated pancreas that we put into the glass vials will now be placed into a 37 degree water bath for approximately 17 minutes. We placed them into a 50 mil conical rack so that they don't float up. Once during the bath, the timer starts and the digestion begins.Once the digestion time is completed, vials are removed, and then they are shaken and disrupted gently. And once the tissue starts falling apart, the vial is then placed on ice. A sterile screen is placed over a 400 mil beaker that contains washing buffer.The pancreas digest is then carefully poured over the screen into the wash buffer. For demonstration purposes, the hood has been turned off and the glass of the hood has been raised so we could see what we're doing. Once the pancreas digest is poured into the beaker, the vials are rinsed with the same washing buffer and the wash buffer placed over the screen.We have a syringe filled with washing buffer. We will use that to forcefully wash the tissue through the screen. The idea is to release any loose eyelets that are unattached to the matrix, but leaving the membranes and the undigested tissue on the screen.And when you're finished washing a tissue, this is what you usually See at the end of the procedure. The Pancreas tissue that's in the bottom of the tube will now be transferred to two 50 mil conical, and the tissue will be washed, the tissue's mixed evenly, and then distributed evenly so that we have a equal pellet volume in each tube. The beaker is now rinsed with more washing buffer.The sides are rinse so that you get all tissue that is entered the beaker again. Then it's equally distributed between each two, both 50 mil. Conical then are topped off with wash buffer.The tissue is inverted several times and then placed into a centrifuge. This is a quick spin. The centrifuge is allowed to reach a thousand RPM and then is turned off.The tissue at this time is very S eyelets are very sensitive, and you don't want to over compact them. The tubes are removed and we will do this wash step three more times. This is the end of the third wash.The components of the 50 mil conical tube are transferred to the 15 conical tube and centrifuge for a thousand RP M and then stopped. The supernat will be removed and the first part of the FI Call will be started. We are at the step where we Have our pelleted di pancreas digest.We'll resuspend that tissue so it's nice and loose to the pellets. We'll add the first density gradient, which is 1 1 0 8. We'll add five mils to each tube.The pellet is then mixed thoroughly in each of the gradients. It's important that you get good tissue distribution in your heaviest gradient. Now we will layer two mils of 1 0 9 6 density.We will then on top of that, layer two mils of 1 0 6 9 density, and then on top of that 1 0 3 7 density. It's important while you're layering the densities that you don't mix. Then all the layers have been added to the tube.And you can see this is the top of 1 1 0 8, the top of 9 1 0 9 6, the top of 1 0 6 9 and the top of 1 0 3 7. And you could see the tissue is already starting to move to its proper density even without centri fusion. The fial gradients are brought up to speed at 1800 RPM for 15 minutes, and remember to keep the brake off after the tubes are removed from the centrifuge.You can see between the 0 0 9 6 and 0 0 7 layer, there's a nice band of eyelet tissue Pablo's. Now removing the 0 3 7 layer to remove any tissue debris and DNA that might have been released during the processing. Using a sterile plastic pasture pipette, he will now remove the islet layer and transfer them to a 50 mil conical containing 25 mils of wash buffer.You want do this step quickly because fol is toxic to eyelets. We actually removed the entire 0 6 9 and 0 9 6 layer. Pavo cuts the top of the plastic pasture pipette and rinses it with wash buffer.The eyelets are then centrifuge like we centrifuge previously. Centrifuge is brought up to a Thousand RPM and then we stop it and We aspirate off. The supernatant eyelets will be washed three times with wash media.So we just completed the eyelid Isolation process we took from the digest to the fol gradient to the purified eyelets after our third wash with wash buffer, we resuspended the eyelets in our RPMI media and we played it in a tissue culture plate. These eyelets are now ready for in vitro assays or in vivo preps like eyelet Transplant.

小鼠胰岛隔离

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

为极化上皮细胞注射质粒的小GTP酶的功能分析

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and 
between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.
The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.

Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

哺乳动物细胞培养，使用一种新的多层瓶中的比例增加

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

测定蛋白酶体降解的植物无细胞系统

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

植物根系的轻质板材荧光显微镜生长在一个凝胶表面

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...