Name: Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry (Video) | JoVE Journal
Uploaded: 2010-07-08T17:15:00.000+00:00
Duration: 11 min 37 s
Description: Learn to measure calpain activity in fixed and living cells using flow cytometry with fluorescent substrates. This method enables researchers to study protease function in leukemia and lymphoma research.

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5 minutes

Supported by grants from the Canadian Institutes of Health Research, The Leukemia and Lymphoma Society of Canada and the Lymphoma Foundation of Canada.

Prepare Reagents
Note: All PBS contains Ca2+ and Mg2+
<ol>
 <li>Detection Reagent 
 Add 20μM 7-amino-4-chloromethyl coumarin, t-BOC-Leucine-methionine amide (BOC-LM-CMAC) to room temperature PBS. Each cell suspension will require 2mL detection reagent.</li>
 <li>1% Paraformaldehyde (Fixed Cells) 
 Dilute 4% paraformaldehyde stock solution in room temperature PBS. Aliquot 1mL 1% paraformaldehyde to each FACS tube. Three FACS tubes are required for each experimental condition (ex. 32Dkit, 32Dkit + PD150606, 32Dkit + PD98059 need 9 FACS tubes)</li>
 <li>PD150606 (calpain inhibitor) 
 Reconstitute PD150606 in DMSO to a final concentration of 100mM. Protect this reagent and all cells treated with it from light for the duration of this experiment.</li>
 <li>PD98059 (MEK1 inhibitor) 
 Reconstitute PD98059 in DMSO to a final concentration of 50mM.</li>
</ol>
Prepare Cells
<ol>
 <li>Grow 32Dkit cells at 5 x 105 to 1 x 106 cells per mL in Opti-MEM media supplemented with 5% fetal bovine serum, WEHI-3 supernatant as a source of IL-3 (or any other source for IL-3 such as recombinant protein), 0.1% 2-mercaptoethanol and antibiotics.</li>
 <li>Collect 12 x 106 32Dkit cells into three 15mL Falcon tubes (4 x 106 cells per tube).</li>
 <li>Pellet the cells at 1000 RPM for 5 minutes at 15°C.</li>
 <li>Aspirate the supernatant. Resuspend the cells in 2mL room temperature PBS.</li>
 <li>Treat one cell suspension with 50μM PD150606. Ensure the sample is protected from light by covering the Falcon tube with aluminum foil. Vortex briefly then incubate for twenty to thirty minutes at room temperature in the dark.</li>
 <li>Treat the second cell suspension with 20μM PD98059. Plate the cells in a 35mm tissue culture dish and incubate for 2 hours at 37°C.</li>
</ol>
Calpain Assay in Fixed Cells
<ol>
 <li>While the samples are incubating with PD150606, begin the calpain assay on the untreated samples. Begin by adding 2mL detection reagent to each cell suspension. Immediately add 1mL of the cell suspension/detection reagent mixture to the previously prepared FACS tubes with 1% paraformaldehyde (0 minutes time point).</li>
 <li>Incubate cell suspensions with detection reagent for 5 minutes at room temperature. Then add 1mL of the cell suspension to a FACS tube with 1mL 1% paraformaldehyde.</li>
 <li>Continue incubating the cell suspensions with detection mixture for an additional 5 minutes at room temperature. Then add 1mL of the cell suspension to a FACS tube with 1mL 1% paraformaldehyde.</li>
 <li>Repeat calpain assay on 32Dkit cells that have been treated with inhibitors.</li>
 <li>Fix cells overnight in the dark at 4°C.</li>
 <li>The next day pellet the cells at 1800 RPM for 5 minutes at 15°C.</li>
 <li>Aspirate the supernatant. Resuspend the cells in 750uL PBS. Place cells on ice and keep in the dark.</li>
</ol>
Acquire Data for Fixed Cells
<ol>
 <li>Analyze the cells using an LSR II (BD Bioscience). Filters are set for the Lightwave Xcyte (UV) laser, excitation line 355nm and laser power 25mW, emission of 405 and 450 nm for substrate and product respectively. Band pass filters for substrate and product are 405/20 and 450/50. Long pass filters for substrate and product are 405 and 450. The PMT voltage was set on 350-375 for substrate and 325-350 for the product.</li>
 <li>Set-up a BD FACSDiva experiment with the following parameters: forward scatter; side scatter; Indo-1 Blue (log); Indo-1 Violet (log). On the worksheet set up the following graphs: forward scatter vs. side scatter dot plot with a gate on live cells (Figure 1a), Indo-1 Blue histogram, Indo-1 Violet histogram, Indo-1 Blue vs. Indo-1 Violet dot plot.</li>
 <li>Calibrate the LSR II with AlignFlow and AlignFlow plus fluorescent beads (Invitrogen). The PMT voltage should be adjusted to place the peak of the bead fluorescence histogram in the same channel prior to every experiment.</li>
 <li>Begin acquiring data using the 32Dkit cells at the 0 minute time point. Adjust parameter voltages as necessary. Acquire and record 10,000 events per sample.</li>
 <li>Repeat acquisition for each sample, rinsing the machine with FACS buffer between each tube.</li>
 <li>Export the data. Ensure LSR II is cleaned according to institute guidelines.</li>
</ol>
Calpain Assay in Live Cells
<ol>
 <li>Prepare the cell suspensions as above, with no PD150606. Bring the samples to the LSR II and set up filters and parameters as above. Set up the worksheet as above. Include a large dot plot showing Indo-1 Blue vs. time. Set the program to acquire the maximum number of events (no stop gate).</li>
 <li>Add 50μM PD150606 to one cell suspension of 32Dkit cells. Keep the sample in the dark and incubate at room temperature for 20-30 minutes.</li>
 <li>Begin acquiring data on the 32Dkit cell suspension with no PD150606. Use a low flow rate of 200-300 events per second. After 30 seconds to 1 minute remove the tube from the machine. Add 20μM BOC-LM-CMAC to the cells. Vortex briefly then continue acquiring the data.</li>
 <li>Acquire data for 10-20 minutes or until the calpain activity plateaus.</li>
 <li>Repeat steps 3 and 4 with 32Dkit cells treated with PD150606.</li>
 <li>Export the data. Ensure LSR II is cleaned according to institute guidelines.</li>
</ol>
Calpain Assay in Complex (Mixed) Cell Populations
*This experiment will identify 32Dkit cells in a cell suspension harvested from a mouse spleen.
<ol>
 <li>Harvest the spleen from each mouse. Lyse the red blood cell with NH4Cl for 10 minutes at room temperature.</li>
 <li>Prepare single cell suspensions in 2mL PBS.</li>
 <li>Divide each cell suspension in two. Treat 1 cell suspension with 50μM PD150606 for 20-30 minutes at room temperature in the dark.</li>
 <li>Proceed as above for the Calpain Assay in Fixed Cells (Steps 1-6)</li>
 <li>Aspirate the supernatant. Wash the cells in 500μL PBS. Aspirate the supernatant.</li>
 <li>Resuspend the cells in 200μL staining buffer with 2% BSA. Incubate for 15 minutes at room temperature.</li>
 <li>Prepare the primary antibody (FITC anti-mouse CD117). The antibody should be used at a 1:200 dilution in staining buffer. Ensure the antibody is kept in the dark.</li>
 <li>Add 200μL primary antibody to the cells. Incubate for 30 minutes at room temperature in the dark.</li>
 <li>Add 400μL PBS to increase the volume for flow cytometry. Place the cells on ice and keep in the dark.</li>
 <li>Proceed as described in Acquire Data for Fixed Cells. Add FITC to the parameters and include a histogram for FITC on the worksheet.</li>
</ol>
Analysis and Representative Results
<ol>
 <li>Analyze data using FlowJo (Treestar). Gate on live cells based on forward and side scatter profile (Figure 1a). Determine the mean fluorescence for Indo-Blue from the viable cell gate (Figure 1b).</li>
 <li>a) For the Mixed Cell Population
 <ul>
 <li>use FITC fluorescence on histograms and dot plots to identify the 32Dkit population</li>
 <li>Determine the mean fluorescence for Indo-Blue of the 32Dkit population</li>
 </ul>
 </li>
 <li>Use Microsoft Excel to graph the mean fluorescence (Figure 2a). Calculate the slope of the line between 5 and 10 minutes. Graph the slope as a bar graph to determine calpain activity in the cells (Figure 2b).</li>
</ol>
<img src="/files/ftp_upload/2050/2050fig1a.jpg" alt="Flow cytometry diagram; FSC/SSC plot showing live cell population distribution analysis." data-altupdatedat="1788235205000" data-alt="Figure 1a"> 
 Figure 1a. Determining live cells based on forward and side scatter profile.
<img src="/files/ftp_upload/2050/2050fig1b.jpg" alt="Flow cytometry results, Indo-1 stained cell analysis, distribution histogram, data comparison." data-altupdatedat="1788235205000" data-alt="Figure 1b"> 
 Figure 1b. Mean fluorescence change over 10 minute time course.
<img src="/files/ftp_upload/2050/2050fig2a.jpg" alt="Line graph showing biochemical response over time with and without PD150606 inhibitor." data-altupdatedat="1788235205000" data-alt="Figure 2a"> 
 Figure 2a. Representative graph of mean fluorescence.
<img src="/files/ftp_upload/2050/2050fig2b.jpg" alt="Calpain activity bar graph, 32Dkit effects with inhibitors PD150606, PD98059, experiment results." data-altupdatedat="1788235205000" data-alt="Figure 2b"> 
 Figure 2b. Calpain activity in 32Dkit cells with inhibitors.

<ol><li>Goll, D. E., Thompson, V. F., Li, H., Wei, W., Cong, J. The Calpain System. Physiol. Rev. 83, 731-801 (2003).</li>
<li>Niapour, M., Berger, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+Cytometric+Measurement+of+Calpain+Activity+in+Living+Cells%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Flow Cytometric Measurement of Calpain Activity in Living Cells.</a> Cytometry A. 71A, 475-485 (2007).</li>
<li>Leloup, L., Daury, L., Mazéres, G., Cottin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Involvement+of+the+ERK%2FMAP+kinase+signalling+pathway+in+milli-calpain+activation+and+myogenic+cell+migration%5BTitle%5D)+AND+%22The+International+Journal+of+Biochemistry+and+Cell+Biology%22%5BJournal%5D)">Involvement of the ERK/MAP kinase signalling pathway in milli-calpain activation and myogenic cell migration.</a> The International Journal of Biochemistry and Cell Biology. 39, 1177-1189 (2007).</li>
</ol>

No conflicts of interest declared.

The calpain activity in fixed and living 32Dkit cells has been determined in this video protocol. Treatment of the cell line with the calpain inhibitor PD150606 resulted in complete inhibition of calpain activity in the cells. In addition, treatment with the MEK1 inhibitor PD98059 resulted in complete inhibition of calpain activity in the cells. ERK is a major activator of calpain-2 3. These results suggest that calpain-2 activity is predominant in the 32Dkit cells. Current work in our laboratory is investigat...

<table><tbody><tr><td>PBS BOC-LM-CMAC 4% paraformaldehyde</td><td>Invitrogen</td><td></td><td>&lt;br/&gt;&lt;br/&gt;Dissolve 4g paraformaldehyde in 100mL PBS. In fume hood, heat sample with stirring until clear.&lt;br/&gt;Let cool then store at 4&amp;#x00B0;C</td></tr><tr><td>PD150606</td><td>Invitrogen</td><td></td><td>Use freshly reconstituted PD150606 for maximal calpain inhibition.</td></tr><tr><td>PD98059</td><td>Cell Signaling Technology</td><td></td><td></td></tr><tr><td>AlignFlow, AlignFlow plus fluorescent beads FACS tubes</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>LSR II</td><td>BD Biosciences</td><td></td><td>&lt;br/&gt;Supernatant of WEHI-3 cells is used as a source of IL-3 in these experiments. Each collection of WEHI-3 supernatant is titrated to determine the optimal concentration used for 32Dkit cell growth. </td></tr></tbody></table>

measuring calpain activity in fixed and living cells by flow cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Cal pains are ubiquitous, intracellular calcium sensitive, neutral cysteine proteases that play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis, and cell cycle progression to measure calpain activity in fixed and live cells, the fluorescent substrate BUC, LMC MAC is added. Buc LMC MAC is specifically cleaved by calpain, resulting in an increase in fluorescence. The change in fluorescence is monitored by flow cytometry, which reveals increased cal pain activity in 30 2D kit cells as compared to normal cytes.Hi, I'm Christina Farr from the laboratory of Dr.Stewart Berger at the University of Toronto in the Department of Immunology and the University Health Network. Today I'm gonna show you how to measure cal pain activity in fixed and live cells using a flow cytometry based assay. We use this assay in our lab to look at cal pain activity in leukemia and lymphoma cells.So let's get started. Prior to beginning experiments, prepare the needed reagents including detection reagent 1%PARAFORM aldehyde cal pain inhibitor PD 1 50 0 6 0 6, and MEC one inhibitor PD 9 8 0 5 9. For details on preparation of these reagents, please see the accompanying written protocol Eloqua one milliliter of 1%para formaldehyde to each fax tube.Three fax tubes are required for each experimental condition for the fixed cells shown in this video. Three conditions are tested, so nine fax tubes are needed. Begin with 32 dki cells at 0.5 to one times 10 of the six cells per milliliter grown in optimum media.Supplemented with fetal bovine serum IL three tumor capto ethanol and antibiotics. Transfer four times 10 to the six cells into each of three 15 milliliter falcon tubes. Place the tubes in the centrifuge and pellet them by spinning at 225 Gs for five minutes at 15 degrees Celsius.Following the centrifuge agent aspirate the snat resuspend the cells in two milliliters of room temperature PBS treat one cell suspension with 50 micromolar cal pain inhibitor PD 1 50 0 6 0 6. Ensure the sample is protected from light by covering the tube with aluminum foil. Vortex Briefly then incubate for 20 to 30 minutes at room temperature in the dark.Treat the second cell suspension with 20 micromolar me one inhibitor PD 9 0 5 9. Plate the cells in a 35 millimeter tissue culture dish and incubate them for two hours at 37 degrees Celsius while the samples are incubating. Begin the cal Calpain assay on the untreated samples.Begin by adding two milliliters of detection reagent to the cell suspension. Immediately add one milliliter of the cell suspension detection reagent mixture to the previously prepared fax tubes to generate the T equals zero sample. Incubate the cell suspensions with detection reagent for five minutes.At room temperature, add one milliliter of the cell suspension detection reagent mixture to the previously prepared fax tube with one milliliter of 1%paraform aldehyde to generate the T equals five sample. Continue incubating the remaining cell suspensions with detection mixture for an additional five minutes at room temperature. After five minutes transfer another one milliliter of the cell suspension to a prepared fax tube with one milliliter of 1%Paraform aldehyde.This is the T equals 10 minute time point. Next, repeat the cal pain assay on 30 2D kit cells that have been treated with inhibitors. Cover the rack with aluminum foil.Fix the cells overnight in the dark at four degrees Celsius the next day. Pellet the cells by centrifuging them at 725 Gs for five minutes at 15 degrees Celsius. Following the spin aspirate the supernatant then resuspend the cells in 750 microliters of PBS.Place the cells on ice and keep them in the dark until it is time to perform flow cytometry.Here. Data acquisition is demonstrated for fixed cells. Perform flow cytometry using an LSR two.Set the filters for the light wave ex excite or UV laser excitation line at 355 nanometers and the laser power at 25 milliwatts with an emission of 405 and 450 nanometers for substrate and product respectively. The BAM pass filters used for substrate and product should be 405 over 20 and 450. Over 50 longpass filters for substrate and product should be 405 and 450.Set the PMT voltage to 350 to 375 for the substrate and 325 to 350 for the product. Set up a BD fax diva experiment with the following parameters. Forward scatter side scatter Indo one blue using a log scale and one violet.Also using a log scale on the worksheet. Set up the following graphs. A forward scatter versus side scatter plot with a gate on live cells.An Indo one blue histogram, an Indo one violet histogram and an Indo one blue versus Indo one Violet dot plot calibrate the LSR two with a line flow and a line flow plus fluorescent beads. The PMT voltage should be adjusted to place the peak of the bead fluorescent histogram in the same channel prior to every experiment. Begin acquiring data using the 30 2D kit cells at the zero minute time point.Adjust parameter voltages as necessary. A acquire and record 10, 000 events per sample. Rinse the machine with fax buffer and repeat the acquisition for each sample.Export the data following data acquisition. Ensure the LSR two is cleaned according to institute guidelines. Prepare the cell suspensions as before without adding the detection reagent or the cal pain inhibitor.Bring the samples to the LSR two and set up filters and parameters as before. Set up the worksheet as before. Include a large dot plot showing Indo one blue versus time.Set the program to acquire the maximum number events. No stop gate. Add 50 micromolar of cal pain inhibitor to one cell suspension of 32 G kit cells.Keep the sample in the dark and incubate it at room temperature for 20 to 30 minutes while the cells are incubating. Begin acquiring data on the 30 2D kit cell suspension without the cal pain inhibitor. Use a low flow rate of 200 to 300 events per second.After 30 seconds to one minute, remove the tube from the machine. Add 20 micromolar of Bach LMC mac to the cells. This is the detection reagent.It is cleaved specifically by calpain and its cleavage results in an increase in substrate fluorescence vortex. Briefly then continue acquiring the data. Acquire data for 10 to 20 minutes or until a cal pain activity plateaus.Next, repeat the acquisition with 32 gki cells treated with cal pain inhibitor. When finished, export the data for analysis. Begin the mixed cell assay with a cell suspension generated from a mouse spleen.Divide each cell suspension in two. Treat one cell suspension with 50 micromolar of cal pain inhibitor cover with foil and incubate for 20 to 30 minutes at room temperature in the dark. After performing the calpain assay as previously described for fixed cells, cover the tubes with foil and incubate the cells overnight at four degrees Celsius in the dark.The next day, centrifuge at 725 Gs for five minutes at 15 degrees Celsius. Aspirate the supernatant, wash the cells in 500 microliters of PBS and aspirate the supernatant again resuspend the cells in 200 microliters of staining buffer with 2%BSA incubate for 15 minutes at room temperature. Next, prepare the primary antibody here, ZI anti-US.CD one 17 is prepared in staining buffer such that the final dilution is one to 200. Ensure the antibody is kept in the dark. Add 200 microliters of primary antibody to the cells, incubate for 30 minutes at room temperature in the dark.Next, add 400 microliters of PBS to increase the volume for flow cytometry. Place the cells on ice and keep in the dark. Acquire data using the same flow cytometry procedure shown for fixed cells.Add ZI to the parameters and include a histogram for ZI on the worksheet following the acquisition of data on the flow cytometer. Analyze using FlowJo gait on live cells based on forward and inside scatter profile. Determine the mean fluorescence for Indo Blue from the viable cell gate.In this plot, the red line indicates the mean substrate fluorescence at zero minutes and the blue and green lines indicate mean substrate fluorescence at five and 10 minutes. For the mixed cell population, use FXI fluorescence on histograms and dot plots to identify the 30 2D kip population. Determine the mean fluorescence for endo blue of the 30 2D kip population.Finally, use Microsoft Excel to graph the mean fluorescence. Calculate the slope of the line between five and 10 minutes. Graph the slope as a bar graph to determine cal pain activity in the cells.The data shown here indicate that 30 2D cells have high levels of cal pain activity and that cal pain activity can be inhibited by MEK inhibitors. We've just shown you how to measure cal pain activity in fixed and living cells. When doing this technique, it's important to set up the LSR two correctly and to calibrate the machine using fluorescent beads.So that's it. Thanks for watching and good luck with your experiments.

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Learn to measure calpain activity in fixed and living cells using flow cytometry with fluorescent substrates. This method enables researchers to study protease function in leukemia and lymphoma research.

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