Genetic or pharmacological reagents that knock down or inhibit specific calpains can be used to address this problem
Genetic or pharmacological reagents that knock down or inhibit specific calpains can be used to address this problem. Disclosures No conflicts of interest declared. Acknowledgments Supported by grants from your Canadian Institutes of Health Research, The Leukemia and Lymphoma Society of Canada and the Lymphoma Foundation of Canada.. 2 With this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte human population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes. Download video file.(134M, mp4) Protocol Prepare Reagents Notice: All PBS contains Ca2+ and Mg2+ Detection Reagent Put 20M 7-amino-4-chloromethyl coumarin, t-BOC-Leucine-methionine amide (BOC-LM-CMAC) to space temperature PBS. Each cell suspension will require 2mL detection reagent. 1% Paraformaldehyde (Fixed Cells) Dilute 4% paraformaldehyde stock solution in space temp PBS. Aliquot 1mL 1% paraformaldehyde to each FACS tube. Three FACS tubes are IFN alpha-IFNAR-IN-1 hydrochloride required for each experimental condition (ex lover. 32Dkit, 32Dkit + PD150606, 32Dkit + PD98059 need 9 FACS tubes) 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. PD98059 (MEK1 inhibitor) Reconstitute PD98059 in DMSO to a final concentration of 50mM. Prepare Cells Grow 32Dkit cells at 5 x 105 to 1 1 x 106 cells per mL in Opti-MEM press supplemented with 5% fetal bovine serum, WEHI-3 supernatant like a source of IL-3 (or any additional resource for IL-3 such as recombinant protein), 0.1% 2-mercaptoethanol and antibiotics. Collect 12 x 106 32Dkit cells into three 15mL Falcon tubes (4 x 106 cells per tube). Pellet the cells at 1000 RPM for 5 minutes at 15C. Aspirate the supernatant. Resuspend the cells in 2mL space temperature PBS. Treat one cell suspension with 50M PD150606. Ensure the sample is safeguarded from light by covering the Falcon tube with aluminium foil. Vortex briefly then incubate for twenty to thirty minutes at space temperature in the dark. Treat the second cell suspension with 20M PD98059. Plate the cells inside a 35mm cells tradition dish and incubate for 2 hours at 37C. Calpain Assay in Fixed Cells While the samples are incubating with PD150606, begin the calpain assay within 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 moments time point). Incubate cell suspensions with detection reagent for 5 minutes at space temperature. Then add 1mL of the Vegfa cell suspension to a FACS tube with 1mL 1% paraformaldehyde. Continue incubating the cell suspensions with detection mixture for an additional 5 minutes at space temperature. Then add 1mL of the cell suspension to a FACS tube with 1mL 1% paraformaldehyde. Repeat calpain assay on 32Dkit cells that have been treated with inhibitors. Fix cells over night in the dark at 4C. The next day pellet the cells at 1800 RPM for 5 minutes at 15C. Aspirate the supernatant. Resuspend IFN alpha-IFNAR-IN-1 hydrochloride the cells in 750uL PBS. Place cells on snow and keep in the dark. Acquire Data for Fixed Cells Analyze the cells using an LSR II (BD Bioscience). Filters are arranged for the Lightwave Xcyte (UV) laser, excitation collection 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 arranged on 350-375 for substrate and 325-350 for the product. Set-up a BD FACSDiva experiment with IFN alpha-IFNAR-IN-1 hydrochloride the following guidelines: ahead scatter; part scatter; Indo-1 Blue (log); Indo-1 Violet (log). Within the worksheet setup the following graphs: ahead scatter vs. part scatter dot storyline having a gate on live cells (Number 1a), Indo-1 Blue histogram, Indo-1 Violet histogram, Indo-1 Blue vs. Indo-1 Violet dot storyline. Calibrate the LSR II with AlignFlow and AlignFlow plus fluorescent beads (Invitrogen). The.