We would like to thank the clinical team of the University Hospital Giessen-Marburg for the blood collection. This work was funded by the Justus Liebig university.

All protocols described in this manuscript for blood collection, isolation and analysis have been reviewed and approved by the Institutional Review Board at the University of Giessen, Germany. The consent of the patients to include their samples in the study was obtained. All steps for isolation and cell culture are carried out under a biological safety cabinet.
1. Venipuncture
<ol>
	<li>Prepare all equipment needed for blood collection including disinfection spray, sterile swab, blood collection cannula with 80 mm tube and multi-adapter, tourniquet/blood pressure cuff, Monovette 9 mL lithium-heparin. 
	NOTE: EDTA as an anticoagulant is also effective.</li>
	<li>Collect blood from the most appropriate arm vein, usually vena mediana cubiti or vena cephalica.</li>
	<li>Apply a tourniquet/blood pressure cuff with light pressure, around 80 mm/Hg.</li>
	<li>Disinfect gloves and puncture site with disinfectant spray containing alcohol. Allow the disinfected puncture site to air dry.</li>
	<li>The veins protrude due to the pressure of the pressure cuff. Insert the needle (Cannula diameter (outer) 21G / 0.8 mm, length 19 mm) at a 15&#176;-20&#176; angle of the vein attempting to avoid trauma and minimizing probing.</li>
	<li>Take blood with appropriate system, 4 tubes containing 9 mL of blood (more than 7-8 tubes are problematic for one experimenter to isolate properly).</li>
	<li>After blood collection, place the collection tubes in the dark for 5 min to ensure uniform anticoagulation.</li>
</ol>
2. PBMC isolation
<ol>
	<li>Prepare all needed solutions as described below.</li>
	<li>Bring Dulbecco&#39;s balanced salt solution (DPBS; concentration 1x) and lymphocyte isolation medium (1.077 g/mL) to room temperature (20-25 &#176;C).</li>
	<li>Prepare Fetal bovine serum (FBS) at room temperature and keep one sterile 50 mL conical tube with FBS on ice. For each blood sample, 2 mL of FBS is required.</li>
	<li>Store freezing container at 4 &#176;C and precool cryotubes at 4 &#176;C.</li>
	<li>Warm cell culture medium to 37 &#176;C, the medium consists of RPMI 1640 with 50 mL of FBS and penicillin 50 U/mL streptomycin 50 U/mL. This solution can be stored at 3 &#176;C for up to 2 months.</li>
	<li>Add 8 mL of DPBS in sterile 50 mL conical tubes. Add 15 mL of lymphocyte isolation medium in sterile 50 mL conical tube (Medium is light sensitive, add it before starting isolation).</li>
	<li>Add 8 mL of blood to 8 mL of DPBS, and carefully further mix with a 3 mL plastic Pasteur pipette.</li>
	<li>Layer the blood/DPBS mix gently with a 3 mL plastic Pasteur pipette on top of the lymphocyte isolation medium. To apply the first layer to the medium, tilt the tube 20&#176;-30&#176;, which will result in less penetration of the blood-PBS mixture into the medium layer.</li>
	<li>Layer the blood-PBS mixture carefully over the side wall of the tube onto the lymphocyte isolation medium. Use a steady speed to keep the blood flow constant.</li>
	<li>In the next step, bring the tube slowly into an upright position, the remaining blood is carefully layered over the side wall of the tube onto the blood layer.</li>
	<li>Centrifuge for 10 min at 1000 x g at room temperature in a centrifuge with a swinging-bucket rotor with brakes off. After centrifugation, the blood/PBMC mix is separated into four layers. The top layer consists of plasma and platelets, the second layer is the PBMC layer, followed by a lymphocyte isolation medium layer, and, finally, erythrocytes and granulocytes in the bottom layer. The different layers are displayed in Figure 1.</li>
	<li>Remove 2/3rd of the plasma layer with a plastic Pasteur pipette.</li>
	<li>Using a 1 mL pipette, collect the PBMCs on the lymphocyte isolation medium layer taking care not to get any medium in the sample.</li>
	<li>Place the tip 1 mm above the PBMC layer. The PBMC layer should not be punctured, otherwise the medium will flow over the cells. The suction of the pipette pulls the PBMCs to this point so that they can be collected several times at this point. 
	NOTE: To maximize the number of PBMCs collected, at the end of the procedure search for the rest on the surface and try to collect the cells there as well. To stabilize the procedure, the tube can be placed on a surface.</li>
	<li>Transfer the PBMCs step by step to a new 50 mL tube until the layer is completely harvested. Add DPBS up to the 25 mL mark, wash away lymphocyte isolation medium and other residues.</li>
	<li>Centrifuge for 10 min at 100 x g at room temperature with brakes on. Remove the supernatant with a vacuum pump or similar, take care not to damage the cell pellet.</li>
	<li>Resuspend the pellet in 1 mL of DPBS and add up DPBS to the 25 mL mark. Repeat washing once again and then resuspend in medium appropriate for the next steps.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65730/65730fig01.jpg" /> 
Figure 1: Schematic representation of a density gradient centrifugation to illustrate the different layers. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65730/65730fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Cryopreservation
<ol>
	<li>Cool down a freezing container to 4 &#176;C, cool FBS on ice.</li>
	<li>Resuspend PBMC pellet in 1 mL of FBS with a 1 mL pipette, the FBS should be at room temperature.</li>
	<li>Mix DMSO with pre-cooled FBS on ice 1:5, final concentration 20% DMSO then put the FBS: DMSO mixture back on ice. Always prepare the solution fresh.</li>
	<li>Transfer the PBMC cell suspension in FBS to well labelled 2 mL cryotubes. Use one cryotube per collection tube. Adjust the number of cells between 1 x 107 and 5 x 107 per mL. Use an automated cell counter to determine cell count and viability.</li>
	<li>Add 1 mL of FBS: DMSO mixture dropwise with a 1 mL pipette, approximately 1-2 drops per s, into the tube. The dropwise addition leads to continuous and consistent mixing.</li>
	<li>Place tubes in the precooled freezing container. Place the freezing container in a -80 &#176;C freezer for 24 h. The freezing container provides a controlled cooling of -1 &#176;C per min.</li>
	<li>Remove the tubes from the -80 &#176;C freezer. After removal from the freezer store the tubes in the gas phase of liquid nitrogen. Document the place of each sample.</li>
</ol>
4. Thawing
<ol>
	<li>Prepare all needed solutions: Cell culture medium RPMI 1640 with 50 mL of FBS and penicillin 50 U/mL, streptomycin 50 U/mL. This solution can be stored at 3&#176;C for up to 2 months.</li>
	<li>Pre-warm cell culture medium to 37 &#176;C. Add 3 mL of pre-warm cell culture medium into sterile 50 mL conical tubes.</li>
	<li>Remove sample from liquid nitrogen tank. Thaw samples in a water bath at 37 &#176;C for approx. 3.5 min, remove from the water bath as soon as the last ice is melting. A pinhead-sized piece of ice should still be visible in the tube. 
	NOTE: DMSO is harmful to PBMCs work as quickly as possible.</li>
	<li>Remove the cell:FBS:DMSO mix with a 1 mL pipette from the cryotube. Mix the PBMC samples with the cell culture medium in the prepared 50 mL tubes. Wash out the tubes with 5 mL of culture medium in three steps of 2 mL, 2 mL, and 1 mL.</li>
	<li>Transfer the medium into the tubes. This is performed to transfer possible cell residues. Centrifuge for 10 min at 100 x g at room temperature.</li>
	<li>Discard the supernatant and add 1 mL of medium appropriate for planned use. The cells are ready for subsequent experiments. 
	&#8203;NOTE: However, with functional tests on fresh or frozen cells, a resting period in an incubator (typically overnight) is often recommended after lymphocyte isolation medium based isolation or cell thawing.</li>
</ol>
5. Cell culture
<ol>
	<li>After isolation or thawing culture cells overnight at 37 &#176;C in 5% CO2/95% air.</li>
	<li>Resuspend the cells in 1 mL of RPMI medium supplemented with 10% FBS, penicilin 50 U/mL, streptomycin 50 U/mL. For further use there are many possibilities, treat cells as needed for the assays.</li>
	<li>For general storage use sterile 6 well cell culture plates and harvest cells after resting period. Transfer 1 mL of cell suspension with a 1 mL pipette into a well and add 4 mL of cell culture medium. 
	NOTE: The amount of PBMCs isolated varies greatly between individuals, one well with 5 mL of cell culture medium is sufficient for every 8 mL of blood isolated. However, when isolating PBMCs from buffy coats, the quantity of PBMCs is significantly higher than in whole blood samples and the cells should therefore be divided into several wells.</li>
	<li>Let cells rest 24 h in a humidified atmosphere supplemented with 5% CO2 at 37 &#176;C. This incubation is performed for freshly isolated cells, as well as for cryopreserved cells.</li>
</ol>
6. ATP assay
<ol>
	<li>Resuspend thawed PBMCs in 1 mL of RPMI medium supplemented with 10% FBS, penicillin 50 U/mL, streptomycin 50 U/mL.</li>
	<li>Take a sample and determine the cell count, then perform a live-dead discrimination with trypan blue. Take 10 &#181;L from the resuspended cells and mix with 90 &#181;L cell culture medium. Then take 10 &#181;L and mix with 10 &#181;L of trypan blue. Place the cells either in a cell counting chamber or an automatic cell counter and determine the number of live/dead cells.</li>
	<li>Plate 100 &#181;L of cells at a density of 1 x 105 cells/100 &#181;L per well in a 96-well white polystyrene plate.</li>
	<li>Let cells rest for 24 h in a humidified atmosphere supplemented with 5% CO2 at 37 &#176;C.</li>
	<li>Determine the ATP concentrations with an ATP assay.
	<ol>
		<li>Use light emission that occurs when ATP is combined with luciferin. The emitted light can be evaluated with a plate reader. Remove the plates for the incubator to cool down to room temperature for 15 min. Lyse the cells and leave them for 5 min. Then apply monitoring reagent to the cells and measure according to the manufacturer&#39;s instructions. An internal standard is used to determine the ATP level.</li>
	</ol>
	</li>
</ol>
7. High-resolution respirometry
<ol>
	<li>Turn ON the high-resolution oxygraph and let it warm up for 30 min.</li>
	<li>Treat cells according to a protocol described in Figure 1. Prepare all stocks needed as in Table 1.</li>
	<li>Pipette 2.1 mL of respiration buffer (Table 1) into both high-resolution oxygraph chambers and stir the buffer continuously using a magnetic stirring bar present in the chambers (750 rpm) at 37 &#176;C for 30 min until a stable oxygen flux signal of the polarographic oxygen sensor is obtained. 
	NOTE: In the chambers of the oxygraph, the oxygen consumption in real time (flux) and the oxygen saturation of the chamber are measured with the aid of polarographic oxygen electrodes. Background calibration must be performed to avoid background noise and to ensure reliable results.</li>
	<li>Perform an air calibration of the polarographic oxygen sensor according to the manufacturer&#39;s protocols23.</li>
	<li>Resuspend isolated PBMCs in 1 mL of mitochondrial respiration medium (MIR05 the composition is shown in Table 1) and dilute to 8 x 106 cells/mL.</li>
	<li>After air calibration, aspirate the respiration medium from the oxygraph chamber and add 2.1 mL of cell suspension in each camber of the respirometer. If needed during the measurement reoxygenate the chambers (see Open at point h in Figure 2), the oxygen saturation of the chambers should not fall below 100 &#181;M.</li>
	<li>Close the chambers by inserting the stoppers, the chambers are designed to hold 2.0 mL volume. Aspirate the emerging cell suspension.</li>
	<li>Continuously mix the cell suspension at 37 &#176;C with a magnetic stirrer (750 rpm) located in the chamber. Wait for about 20 min until a stable signal is obtained. Determine endogenous respiration ((a) in Figure 2).</li>
	<li>To determine the different complex activities of the respiratory chain, inject the substrates and inhibitors for mitochondrial respiration through the titanium injection ports of the stoppers. Use the following final concentration in the chamber.</li>
	<li>To disrupt cell membranes, add 5 &#181;L of 8.1 mM digitonin, through the titanium injection port of the chamber stopper, to remove naive substrates (b) in Figure 2 while the mitochondrial membranes remain intact.</li>
	<li>Add substrates 2 M glutamate and 800 mM malate through the injection port of the chamber stopper and record respiration until a stable signal is achieved. The signal stabilizes after 2-4 min. 
	NOTE: It is also possible to use additional substrates like pyruvate.</li>
	<li>Add 8 &#181;L of 500 mM adenosine diphosphate (ADP) through the injection port of the chamber stopper and record respiration until a stable signal is achieved. The signal stabilizes after 2-4 min (d) in Figure 2.</li>
	<li>Add 20 &#181;L of 1 M succinate through the injection port of the chamber stopper and record respiration until a stable signal is achieved. The signal stabilizes after 2-4 min (e) in Figure 2.</li>
	<li>Titrate 1 M carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP) stepwise at 0.5 &#181;L up to the point where no further increase occurs. Wait 2-4 min until the signal stabilizes (f) in Figure 2. When there is no further increase in respiration continue with the next step. 
	CAUTION: Be careful when handling FCCP as it has health risks for humans.</li>
	<li>Add 5 &#181;L of 0.1 mM rotenone through the injection port of the chamber stopper and record respiration until a stable signal is achieved. The signal stabilizes after 2-4 min (g) in Figure 2. 
	CAUTION: Be careful when handling rotenone as it has health risks for humans.</li>
	<li>Add 1 &#181;L of 4 mg/mL oligomycin through the injection port of the chamber stopper and record respiration until a stable signal is achieved. The signal stabilizes after 2-4 min (h) in Figure 2. 
	CAUTION: Be careful when handling oligomycin as it is a poison that poses health risks to humans.</li>
	<li>Add 1 &#181;L of 5 mM antimycin A through the injection port of the chamber stopper and record respiration until a stable signal is achieved. The signal stabilizes after 2-4 min (i) in Figure 2. 
	CAUTION: Be careful when handling antimycin A as it is a poison that poses health risks to humans.</li>
	<li>When evaluating the run to exclude the oxygen consumption of enzymes not involved in oxidative phosphorylation, subtract the antimycin A values from all other measured values.</li>
	<li>Add 200 mM N,N,N&#39;,N&#39;-tetramethyl-p-phenylenediamine dihydrochloride (TMPD; electron donator) and 800 mM ascorbate to keep TMPD in a reduced state. Inject the substrates through the injection port of the chamber stopper and record respiration until a stable signal is achieved. The signal stabilizes after 2-4 min (j) in Figure 2.</li>
	<li>TMPD is subject to autooxidation, so subtract the resulting oxygen consumption from the measured value at Complex IV.</li>
	<li>Add NaN3&#160;&#8805; 100 mM through the injection port of the chamber stopper and record respiration until a stable signal is achieved. The signal stabilizes after 2-4 min to inhibit complex IV activity, only TMPD autooxidation remains. 
	CAUTION: Be careful when handling sodium azide as it is a poison that poses health risks to humans.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65730/65730fig02.jpg" /> 
Figure 2: Schematic course of the O2 flux. The schematic course of the oxygen flux is shown. The curve is divided into the different phases after the addition of inhibitors and substrates from a-k. a: endogenous respiration; b: permeabilized cells; c: uncoupled complex I respiration; d: coupled complex I respiration; e: OXPHOS ; f: maximal uncoupled activity of CI and CII ; g: uncoupled respiration of complex II; h: leak respiration; i: residual respiration; j: CIV(U) uncoupled respiration and autooxidation of TMPD; k: autooxidation of TMPD. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65730/65730fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
8. Citrate synthase activity
<ol>
	<li>Measure citrate synthase activity as a separate parameter and use it to normalize the measurements of the high-resolution oxygraph.</li>
	<li>Resuspend isolated PBMC in 1 mL of mitochondrial respiration medium (MIR05) and dilute to 8 x 106 cells/mL.</li>
	<li>Freeze in liquid nitrogen and store at &#8722;80 &#176;C until experiments are conducted or to measure fresh cells.</li>
	<li>Prepare all solutions needed: 0.1 M triethanolamine HCl buffer pH 8.0, 1.0 M Tris-HCl buffer pH=8.1, 10% Triton X-100, 10 mM Oxalacetate in 0.1 M triethanolamine HCl buffer pH 8.0, 1.01 mM DTNB in 1.0 M Tris-HCl buffer pH=8.1, Acetyl-CoA 12.2 mM in double distilled H2O.</li>
	<li>Prepare reaction medium (Table 1) containing 5,5&#39;-dithio-bis-(2-nitrobezoic acid) (DTNB; 0.1 mM), acetyl coenzyme A (0.31 mM), EDTA (50 &#181;M), triethanolamine HCl (5 mM) and Tris HCl (0.1 M).</li>
	<li>Prepare starting reagent (Table 1) with 0.5 mM oxaloacetate dissolved in double distilled H2O.</li>
	<li>Thaw the samples on ice as citrate synthase is unstable if thawed too quickly.</li>
	<li>Add 40 &#181;L of the samples to a 96-well plate on ice before adding 110 &#181;L of reaction medium with a multipipette.</li>
	<li>Warm reaction medium and sample in an incubator to 30 &#176;C for 5 min. Warm starting reagent in a water bath to 30 &#176;C for 5 min.</li>
	<li>Add 50 &#181;L of the starting reagent with a multipipette to each well. Measure absorbance at 30 &#176;C at a wavelength of 412 nm for 20 min via plate reader.</li>
</ol>

PBMC Collection and Cryostorage for Mitochondrial Bioenergetics Analysis

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J., Stone, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biospecimen+science+of+blood+for+peripheral+blood+mononuclear+cell+(PBMC)+functional+applications.">Biospecimen science of blood for peripheral blood mononuclear cell (PBMC) functional applications.</a> Curr Pathobiol Rep. 7, 17-27 (2019).</li><li>Pesta, D., Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+respirometry:+OXPHOS+protocols+for+human+cells+and+permeabilized+fibers+from+small+biopsies+of+human+muscle.">High-resolution respirometry: OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle.</a> Methods Mol Biol. 810, 25-58 (2012).</li><li>Djafarzadeh, S., Jakob, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+respirometry+to+assess+mitochondrial+function+in+permeabilized+and+intact+cells.">High-resolution respirometry to assess mitochondrial function in permeabilized and intact cells.</a> J Vis Exp. (120), e54985 (2017).</li><li>Wang, W., Zhao, F., Ma, X., Perry, G., Zhu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+dysfunction+in+the+pathogenesis+of+Alzheimer&#8217;s+disease:+recent+advances.">Mitochondria dysfunction in the pathogenesis of Alzheimer&#8217;s disease: recent advances.</a> Mol Neurodegener. 15 (1), 30 (2020).</li><li>Chaturvedi, R. K., Flint Beal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+diseases+of+the+brain.">Mitochondrial diseases of the brain.</a> Free Radic Biol Med. 63, 1-29 (2013).</li></ol>

The authors declare no conflict of interest.

This protocol provides a means of isolating and cryopreserving peripheral blood mononuclear cells (PBMCs) from human blood in a manner suitable for bioenergetic analyses. The described method offers the possibility to isolate PBMCs gently and in large quantities, with high viability and sufficient cells for bioenergetic measurements. It has the disadvantage that even with minimal interruptions, long isolations occur, but subsequent cryopreservation allows a time-independent measurement of bioenergetics. With this method,...

Human peripheral blood mononuclear cells (PBMCs) are used for various applications in many scientific fields, including the study of immunological and bioenergetic issues, such as those related to aging processes or degenerative diseases1,2. PBMCs are heterogeneous in composition and consist of lymphocytes (B cells, T cells, and NK cells), monocytes, and dendritic cells. The cells sometimes show great individual differences and variations within a subject, so standardized procedures for handling these cells are required. Important parameters such as viability and purity of the isolation are the basic requirements for its handling and are additionally influenced by environmental factors such as the time of collection, the melatonin level, whether the subject is fasting, and others3,4.
Based on studies on bioenergetics of PBMCs, we describe here a method for the isolation, cryopreservation, and cultivation of PBMCs that is suitable for other methods as well. While muscle biopsy is considered the gold standard for mitochondrial energy metabolism5, the examination of blood cells is a rapid, minimally invasive procedure. In addition to this, more and more studies suggest that the changes in mitochondrial function in aging and Alzheimer&#39;s disease (AD) occur not only in the brain but also in the periphery6,7,8,9,10. The method also allows investigations of other conditions and diseases, including diabetes mellitus and obesity11,12,13. Gene expression patterns in multiple sclerosis patients can be analyzed, or immune function and influences on it in general14,15,16.
PBMCs generally rely on oxidative phosphorylation (OXPHOS) to generate adenosine triphosphate (ATP)17,18. Therefore, PBMCs cover a wide range of applications as surrogates. In previous reports, the energy metabolism of PBMCs has been used to address organ dysfunctions, such as in early heart failure19, septic shock20 or sex-associated differences4 in mitochondrial function. A generalized method for cryopreservation isolation and cultivation of PBMCs would have advantages in the comparability of results obtained at different institutes. There is a great deal of variation in the protocols for each step21,22, the goal of this method is to provide a guideline for bioenergetic measurements in PBMCs.
In this article we describe a method for measuring bioenergetic parameters in PBMCs. We explain the methods for isolating, cryopreserving and measuring bioenergetics of PBMCs from human blood. This method can be used to determine bioenergetic parameters in patients and evaluate them in a clinical context. To apply these measurements, researchers need access to a patient population from which fresh blood samples can be obtained.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 M Triethanolamine-HCl-Buffer (pH = 8,0)</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M Triethanolamine-HCl-Buffer</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 M Tris-HCl-Buffer (pH = 8,1)</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>1.01 mM DTBB</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>10 % Triton X-100</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Oxalacetat</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>14&#8211;20 G sterile blood draw needles Multi Adapter Sarstedt Safety-Multifly</td>
			<td>Sarstedt</td>
			<td>156353_v</td>
			<td></td>
		</tr>
		<tr>
			<td>37% HCl</td>
			<td>Carl Roth GmbH &#38; Co. KG</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol (EtOH)</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl-CoA</td>
			<td>Pancreac Applichem</td>
			<td>A3753</td>
			<td></td>
		</tr>
		<tr>
			<td>ADP</td>
			<td>Sigma-Aldrich</td>
			<td>A5285</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol wipes</td>
			<td></td>
			<td></td>
			<td>&#160;(70% isopropyl alcohol)</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua (bidest.)</td>
			<td>With MilliQ Academic (self-made)</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP-Standard</td>
			<td>Sigma-Aldrich</td>
			<td>6016949</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocoll Seperating Solution</td>
			<td>Biochrom</td>
			<td>6115</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safty cabinet MSC Advantage</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonylcyanid-p-trifluoromethoxy-phenylhydrazon (FCCP)</td>
			<td>Sigma-Aldrich</td>
			<td>C2920</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter TC20 Automated Cell Counter</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Heraeus Megafuge 16 R</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Counting slides, dual chamber for cell counter</td>
			<td>Bio-Rad</td>
			<td>1450016</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotube Cryo.S</td>
			<td>Grainer Bio-One</td>
			<td>126263-2DG</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Sigma-Aldrich</td>
			<td>37008</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxid (DMSO)</td>
			<td>Merck</td>
			<td>102952</td>
			<td></td>
		</tr>
		<tr>
			<td>Disinfection spray</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable gloves latex, rubber, or vinyl.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Distrips (12.5 ml) DistriTips</td>
			<td>Gilson</td>
			<td>F164150</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (DPBS; 10x)</td>
			<td>Gibco (Thermo Scientific)</td>
			<td>15217168</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (EtOH 100%)</td>
			<td>Carl ROTH GmbH &#38; Co. KG</td>
			<td>9065.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr>
			<td>Frezer (-80&#176;C)</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G1626</td>
			<td></td>
		</tr>
		<tr>
			<td>Holder/adapter&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator Midi 40 CO2</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Injection syringe</td>
			<td>Hamilton</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Malate</td>
			<td>Sigma-Aldrich</td>
			<td>M-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>MIR05</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty Freezing Container</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td>10110051</td>
			<td></td>
		</tr>
		<tr>
			<td>Multireader CLARIOstar</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen tank Locator 6 plus</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>Sigma-Aldrich</td>
			<td>O4876</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxalacetate</td>
			<td>Sigma-Aldrich</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygraph-2k</td>
			<td>Orobororus Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>PAA</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes Performance Pipettor 10 &#956;L, 100 &#956;L, 1000 &#956;L</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell-Park. Memorial-Institute-Medium (RPMI-1640)</td>
			<td>Gibco (Thermo Scientific)</td>
			<td>11530586</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharose</td>
			<td>Carl ROTH GmbH &#38; Co. KG</td>
			<td>9286.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinate</td>
			<td>Sigma-Aldrich</td>
			<td>S2378</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylphenylendiamin (TMPD)</td>
			<td>Sigma-Aldrich</td>
			<td>T3134</td>
			<td></td>
		</tr>
		<tr>
			<td>Tourniquet/ Blood pressure cuff</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)amino-methane</td>
			<td>Sigma-Aldrich</td>
			<td>108382</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>108643</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypanblau</td>
			<td>Biochrom</td>
			<td>T6146</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Vaccubrand GmbH &#38; Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ViewPlate-96</td>
			<td>Perkin Elmer</td>
			<td>6005181</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath WNB22</td>
			<td>Memmert GmbH &#38; Co. KG</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

cryopreservation and bioenergetic evaluation of human peripheral blood mononuclear cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Cell viability and number 
To achieve successful isolation and cryopreservation, cell count and viability should be as high as possible. Before and after cryopreservation, the cells are counted, and their viability is determined to ensure the health and quality of the cells. Figure 3 is a representative illustration of PBMCs before and after cryopreservation, cell count and viability hardly differ. This indicates successful isolation and preservation of PBMCs.
<p cl...

Watch this Scientific Journal Video about Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation at JoVE.com

Author Spotlight: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...

cryopreservation-bioenergetic-evaluation-human-peripheral-blood

Research

JoVE Journal

Immunology and Infection

1.1K Views.  Justus-Liebig-University of Giessen. Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

Video: Author Spotlight: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation 

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

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Chapters in this video

Isolation of Mouse Peritoneal Cavity Cells

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Expansion of Human Peripheral Blood γδ T Cells using Zoledronate

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages