The authors acknowledge Anne Charri&#233; at the Radiobiology service (Lyon-Sud hospital) and the Fond National Suisse (FNS) for their financial support.

1. Preparation of Cell Culture Media and Solutions
<ol>
	<li>Preparation of culture media
	<ol>
		<li>Prepare proliferation medium (PM) by supplementing Ham&#39;s F-10 medium with glutamine (2 mM), penicillin/streptomycin (5 &#181;g/mL final), 2% Fetal Calf Serum (FCS) and 2% serum substitute.</li>
		<li>Prepare differentiation medium (DM) by supplementing Dulbecco&#39;s Modified Eagle Medium (DMEM) with glutamine (2 mM), penicillin/streptomycin (5 &#181;g/mL final), and 2% FCS.</li>
	</ol>
	</li>
	<li>Preparation of glucose uptake solutions 
	Caution: Handling of radioactive material is only allowed in a restricted and controlled area by authorized personnel. Material and waste must be handled according to appropriate procedures, guidelines and local legislation.
	<ol>
		<li>To prepare X-Dulbecco&#39;s phosphate-buffered saline (X-DPBS), make a solution of DPBS containing 0.2% (w/v) (final concentration) bovine serum albumin (BSA). Filter the solution through a 0.2 &#181;m filter. Store at 4 &#176;C.</li>
		<li>To prepare cold 2-deoxy-D-glucose (2dG) solution, weigh 16.4 mg of 2dG and solubilize in 10 mL distilled water to obtain a 10 mM solution. Store at 4 &#176;C.</li>
		<li>Add 600 &#181;L of cold 2dG and 6 &#181;L of radiolabeled [3H] 2dG to 5400 &#181;L of X-DPBS to obtain the radiolabeled 2dG (2dG*) solution. 
		NOTE: The final concentration is 1 mM 2dG and labeling is 1 &#181;Ci/mL.
		<ol>
			<li>Set aside a 20 &#181;L aliquot (TC20) of radiolabeled 2dG* solution.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of incubation mixtures
	<ol>
		<li>For the cytochalasin B mixture, add 2 &#181;L of 20 mM cytochalasin B to 2 mL of radiolabeled 2dG* solution. 
		NOTE: Stock solution of cytochalasin B is at 10 mg/mL in dimethyl sulfoxide (DMSO).</li>
		<li>For the DMSO mixture, add 4 &#181;L of DMSO to the remaining 4 mL of radiolabeled 2dG* solution.</li>
	</ol>
	</li>
</ol>
2. Culture of Human Primary Muscle Cells
<ol>
	<li>Seeding of 6-well plates with human muscle satellite cells 
	NOTE: Use in-house (see reference 24 for details) or commercially available human muscle satellite cells from a frozen vial (containing 250,000 cells). The following procedure is given for 250,000 cells in order to obtain one 6-well plate necessary for the measurement of glucose uptake in a single condition.
	<ol>
		<li>Rapidly thaw frozen vials of in-house24 or commercial preparations of human muscle satellite cells in pre-warmed water (37 &#176;C) until only a small ice block remains in the vial.</li>
		<li>Pour directly into a 50 mL plastic tube containing 10 mL of pre-warmed (37 &#176;C) PM.</li>
		<li>Centrifuge for 5 min at 500 x g and discard the supernatant.</li>
		<li>Gently resuspend the cell pellet with 18 mL of pre-warmed PM (to obtain 42,000 cells per 3 mL of medium). Distribute 3 mL in each well of a 6-well plate (9.6 cm2). 
		NOTE: The six individual wells of a plate are required to perform a duplicate measurement of glucose uptake for the following conditions: passive transport inhibition (wells 1 and 2), basal rate (wells 3 and 4) and insulin stimulated rate (wells 5 and 6). Repeat as many six-well plates as distinct treatments are needed.</li>
		<li>Incubate in standard culture conditions (37 &#176;C, 5% CO2) until cells reach 90% confluence. 
		NOTE: This step takes between 48 - 72 h depending on the cell batch. Do not change medium during this step.</li>
	</ol>
	</li>
	<li>Differentiation of muscle cells
	<ol>
		<li>Remove PM (after 48-72 h) and replace with pre-warmed DM (3 mL per well). Incubate at 37 &#176;C, 5% CO2. 
		NOTE: Differentiation takes five days to reach a stable state where cells are aligned and polynucleated. Typically, the primary myotubes are cultured in a 1 g/L glucose medium. Therefore, to avoid glucose depletion during culture, fill the plate with 3 mL medium to ensure that enough glucose substrate is available for the cells at all time.</li>
		<li>Replace DM medium every two days. 
		NOTE: From this point, myotubes are stable for up to 7 days without any significant change and glucose uptake measurement can be performed at any time.</li>
	</ol>
	</li>
	<li>Muscle cells treatment (optional) 
	NOTE: Primary myotubes can be treated for several days to induce modification (drug test, inhibitors/activators of signaling pathway, etc.) before insulin stimulation and glucose uptake measurements. Muscle cells can be submitted to any treatment that may have an impact on insulin sensitivity, and glucose uptake measurement will quantify this impact. For example, incubation of muscle cells with the saturated fatty acid palmitate promotes insulin resistance, and cells display reduced insulin stimulated glucose uptake.
	<ol>
		<li>Prepare 12 mL of DM supplemented with 10% BSA (fatty acid free) and 0.5 mL palmitate (PALM). Prepare 12 mL of DM supplemented with 10% BSA (fatty acid free) only.</li>
		<li>Prepare two 6-well plates with human primary myotubes, and culture them as described in sections 2.1 and 2.2 (with 5 days of differentiation).</li>
		<li>On day 5, wash each well with 2 mL of PBS. To one plate, add 2 mL of DM containing PALM. To the other plate add 2 mL of BSA only containing DM.</li>
		<li>Incubate for 48 h at 37 &#176;C, 5% CO2.</li>
	</ol>
	</li>
</ol>
3. Insulin Stimulation
<ol>
	<li>Wash differentiated muscle cells twice with 2 mL PBS.</li>
	<li>Remove PBS carefully and incubate with 3 mL of DM without FCS for 3 h (37 &#176;C, 5% CO2) for serum depletion.</li>
	<li>Replace media in all wells with 3 mL of DM without FCS. Add 100 nM insulin to wells 5 and 6.</li>
	<li>Incubate human myotubes culture for 1 h (37 &#176;C, 5% CO2).</li>
</ol>
4. Glucose Uptake
<ol>
	<li>After 1 h of insulin stimulation, wash wells twice with X-DPBS (1 mL per wash).</li>
	<li>Add 1 mL of cytochalasin B mixture to wells 1 and 2, and 1 mL of DMSO mixture to wells 3 - 6. Incubate for 15 min (37 &#176;C, 5% CO2). At the end of the incubation, immediately place the plate on ice.</li>
</ol>
5. Cell Lysis
<ol>
	<li>Wash the cells twice with 1 mL of ice cold PBS.</li>
	<li>Lyse the cells in each well with 600 &#181;L of 50 mM NaOH. Incubate on ice for 5 min and mix gently with slow orbital rotation. 
	NOTE: If the lysate is too viscous, dilute with up to 1.5 mL NaOH.</li>
	<li>Using a pipette, resuspend and collect the cell lysate.</li>
</ol>
6. Determination of Radiolabeled Glucose
<ol>
	<li>Put 400 &#181;L of each cell lysate in a liquid scintillation counting vial. Prepare a negative control vial with 400 &#181;L of 50 mM NaOH, and a positive control vial with 20 &#181;L of TC20 (from step 1.2.3.1).</li>
	<li>Add 4 mL of liquid scintillation solution to each vial. Close the cap and mix each vial thoroughly (1-2 s).</li>
	<li>Insert each vial in a liquid scintillation counter and measure the radioactivity according to the manufacturer&#39;s instruction. Record counts per min (CPM) for each scintillation vial for 10 min. 
	NOTE: CPM = &#34;disintegrations per minute&#34; x &#34;counting efficiency&#34;.</li>
</ol>
7. Rate of Glucose Uptake
<ol>
	<li>Use the remaining lysate (200 &#181;L; from step 5.2) to measure the protein concentration. Determine the protein concentration of each cell lysate using Bradford25 or an equivalent method. Calculate total protein quantity (Q) in mg for each well.</li>
	<li>To obtain TC1 (the value for 1 &#181;L of radiolabeled 2dG*), divide the CPM value of TC20 by 20.</li>
	<li>For each vial, calculate the rate of glucose uptake as follows: 
	<img alt="Equation" src="/files/ftp_upload/55743/55743eq1.jpg" /> 
	NOTE: R is measured in pmol/mg/min. Mean of R for wells 1 - 2 gives passive transport rate, Rp. Mean of R for wells 3-4 gives basal total transport rate, Rbt. Mean of R for wells 5-6 gives insulin stimulated total transport rate, Rit.
	<ol>
		<li>Calculate the basal active transport rate (Rba) as follows: 
		<img alt="Equation" src="/files/ftp_upload/55743/55743eq2.jpg" /></li>
		<li>Calculate the insulin stimulated active transport rate (Ria) as follows: 
		<img alt="Equation" src="/files/ftp_upload/55743/55743eq3.jpg" /> 
		NOTE: In insulin responsive cells like myotubes, glucose uptake rates are usually represented by three values: Rba, Ria, and the fold insulin stimulation as Ria/Rba.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Glucose uptake is a key biological measurement for testing activators or inhibitors on cell culture and how they impact glucose use, and the ability of the cell to respond to insulin. The method described here has been shown to be quick and reliable and has been widely used in many studies using primary myotubes from healthy subjects and/or metabolically affected patients6,7,10,12,<s...

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. This insulin responsive tissue is the primary site of the glucose uptake that is triggered by insulin stimulation1.
In type 2 diabetes, insulin resistance is observed in several tissues, including skeletal muscle, and leads to above normal blood glucose concentration. Thus, it is of major relevance to determine the level of insulin sensitivity of this tissue and its cells, whether the aim is to characterize a defect in a subject, or to evaluate the efficiency of a treatment intending to improve it. In human or animal subjects, the gold standard technique to assess insulin sensitivity is the hyperinsulinemic-euglycemic clamp. Introduced by DeFronzo in 19792 and modified since3,4 then, the method allows to quantify whole body and tissues insulin responsiveness measured as the rate of glucose to be perfused under insulin stimulation to maintain normal blood glucose concentration.
Insulin sensitivity exploration can also be performed at the cell level using in vitro muscle models, and measurement of glucose uptake rates remains an efficient and reliable tool to quantify the biological response of the cell to insulin stimulation5,6,7. Indeed, glucose uptake measurement quantifies the cell biological response to insulin stimulation, from the binding of insulin to its receptor to translocation of GLUT4 enriched vesicles, and including intracellular signaling and phosphorylation cascades8.
This is of major interest with human samples, as differentiated myotubes maintain many features of the donor phenotype, including metabolic properties and disorders observed in the patient9,10,11,12. The myotubes displays structural, metabolic and phenotypic similarities to the skeletal muscle13,14, including the expression of glucose transporters15 and the cellular insulin signaling machinery16. Thus, measurement of the glucose uptake in primary myotubes is of relevance to characterizing the muscle phenotype of a donor, or investigating the effect of an intervention (drug, nutrition, or physical activity) on the insulin sensitivity in the muscle cell.
The measurement of glucose uptake on cultured myotubes also is a reliable tool when performing experiments that modify insulin sensitivity17,18. The in vitro model is suitable for the test of any compounds that could improve insulin responsiveness, or could prevent or reverse acquired or induced insulin resistance19,20,21,22,23.
Here we describe a detailed protocol to culture and differentiate human myotubes and to measure cell glucose uptake rates. The method is applicable to any source of human muscle precursor cells, whether they come from in-lab preparations, collaboration, or commercially available suppliers. Immortalized muscle cell lines, like C2C12 and L6, respectively from mouse and rat origin, can also be used for glucose uptake measurement with this protocol7.
We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H]2dG. The use of a labeled glucose analog allows accurate determination of glucose entry with reduced starting material, a common condition when working with primary cells. The modified glucose molecule is unable to enter metabolic pathways, and thus, accumulates within the cell, allowing reliable quantification via total cell radioactivity. Experimental conditions include the use of a glucose transport inhibitor (cytochalasin B), and measurements are performed with and without insulin. This combination allows the determination of glucose active entry rates, as well as the calculation of fold change for the insulin response index. The method is presented with one dose of insulin during a single incubation time, but the protocol can easily be modified for dose response or time course experiments12.

<table border="0" cellpadding="0" cellspacing="0" width="868">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Human primary muscle cell</td>
			<td style="width:171px;">In house preparation from human skeletal muscle biopsies</td>
			<td style="width:180px;">In house preparation from human skeletal muscle biopsies</td>
			<td style="width:257px;">If not available, use commercial source</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Human primary muscle cell</td>
			<td style="width:171px;">Promocell</td>
			<td>C-12530</td>
			<td style="width:257px;">Should be cultured with associated media C23060 and C23061</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">6-well plate</td>
			<td style="width:171px;">Corning</td>
			<td style="width:180px;">356400</td>
			<td style="width:257px;">BioCoat Collagen I Multiwell Plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Ham&#39;s F10</td>
			<td style="width:171px;">Dutscher</td>
			<td style="width:180px;">L0145-500</td>
			<td style="width:257px;">1 g/l glucose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Glutamine</td>
			<td style="width:171px;">Dutscher</td>
			<td style="width:180px;">X0551-100</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">penicilin/streptomycin 100x</td>
			<td style="width:171px;">Thermo fisher scientific</td>
			<td style="width:180px;">15140122</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Serum substitute UltroserG</td>
			<td style="width:171px;">Pall France</td>
			<td style="width:180px;">15950.017</td>
			<td style="width:257px;">serum substitute in text</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">DMEM low glucose</td>
			<td style="width:171px;">Dutscher</td>
			<td style="width:180px;">L0064-500</td>
			<td style="width:257px;">1 g/l glucose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Fetal Calf Serum</td>
			<td style="width:171px;">Eurobio</td>
			<td style="width:180px;">CVFSVF00-01</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td style="width:171px;">Dutscher</td>
			<td style="width:180px;">L0625-500</td>
			<td style="width:257px;">Contains Mg2+ (0.5 mM) and Ca2+ (0.9 mM)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Insulin solution human</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:180px;">I9278</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">2-deoxy-D-glucose&#160;</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:180px;">D6134</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Albumin bovine</td>
			<td style="width:171px;">euromedex</td>
			<td style="width:180px;">04-100-812-E</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">fatty acid-free BSA</td>
			<td style="width:171px;">Roche</td>
			<td style="width:180px;">10,775,835,001</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">palmitate</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:180px;">P0500</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Deoxy-D-glucose, 2-[1,2-3H (N)]</td>
			<td style="width:171px;">PerkinElmer</td>
			<td style="width:180px;">NET328A001MC</td>
			<td style="width:257px;">Specific Activity: 5-10Ci (185-370GBq)/mmol, 1mCi (37MBq</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Cytochalasin B</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:180px;">c2743</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PICO PRIAS VIAL 6ml</td>
			<td style="width:171px;">PerkinElmer</td>
			<td style="width:180px;">6000192</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">ultima gold MW CA&#160;</td>
			<td style="width:171px;">PerkinElmer</td>
			<td>6013159</td>
			<td style="width:257px;">scintillation liquid</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">b&#234;ta counter&#160;</td>
			<td style="width:171px;">PerkinElmer</td>
			<td style="width:180px;">2900TR</td>
			<td style="width:257px;"></td>
		</tr>
	</tbody>
</table>

glucose uptake measurement and response to insulin stimulation in in vitro cultured human primary myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

On day 3, myoblasts reach confluence (Figure 1A). The myoblasts at this stage are typically mononucleated. Medium was changed and on day 8, differentiation was completed (Figure 1B) (protocol section 2). After 5 days of differentiation, myotubes are aligned and typically polynucleated. Human primary myotubes were subjected to a palmitate or a BSA-only treatment before glucose uptake rate measurement. Cells were incubated for 48 h...

Watch this Scientific Journal Video about Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes at JoVE.com

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes | Biology | Video

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

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19.7K Views.  University of Lyon. In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Video: Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 &#176;C instead of the conventional 70-100 &#176;C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other &#39;large&#39; proteins like BRCA2 should be readily achievable.

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...