User:Nicole M Paterson/Notebook/General Methods/Entry Base
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B Cell Activation/Detection of Phosphorylation Differentiation Markers and Apoptosis From Emily Goren 2010, revised 2014 from Flow Cytometry, Humana Press Intracellular phosphorylation staining Day 0 Euthanize mouse, remove both fibulas by cutting the ligaments at the knee and twisting the bone at the hip until intact bone is removed. Clean off bone with sterile gauze. Cut off top and bottom of bone ends with sterilized scissors. With large gauge needle, wash out bone marrow cells with several ml B cell media into 50ml Falcon tube with nylon mesh filter attached to the top. Spin at 1000 RPM for 10min at RT. Remove transparent fluid, add 1ml ACK lysis buffer, incubate 6min. Stop reaction with 20-25ml B cell media. Spin at 1000 RPM for 10min at RT, remove fluid add 5ml B cell media + cytokines (m-SCF 10ng/ml), and count cells with hemocytometer. Dilute to 1.0 x 106 cells/ml and plate. Add contents to TC treated petri dish, adding B cell media + cytokines to total volume of 10ml. Incubate o/n at 37°C 5% CO2. Detection of phosphorylation of SYK and B cell Maturity Aliquot 100,000 cells/FACS tube (about 100µl) Add CD-19 APC 1:250 to cells, incubate 20min RT in dark, keep in B cell media. This is the B cell marker, need one for single stain, one for SYK dual stain. Also need additional two unstained control, seven tubes total. Tube 1: Unstained Cells Tube 2: CD19-APC only Tube 3: SYK-PE only Tube 4, 5, 6, 7: SYK-PE/CD19 APC Cells (4 for 4 time points) Incubate cells in 100µl media for 10min at 37°C 5% CO2 Preheat Cytofix. Activate B cells by adding 2µl Fab/tube. Keep at 37°C add 210µl warm cytofix at 0, 1, 2, 4, 8 minutes (five tubes plus two controls, total of seven) vortex after adding. Note: 2 minutes has been found in the past to yield best results and can serve as a trial time point. Incubate for 8-15min at 37°C 5% CO2 Wash cells in several ml FACS Buffer, spin at 1000 RPM for 10min at RT. Vortex and resuspend cells in 200µl ice cold Phosphorylation Perm Buffer from -20°C freezer. Incubate 30min in the refrigerator in the dark. Wash cells with 3ml BSA stain buffer, spin at 1000 RPM for 10min at RT. Re-suspend in 0.1ml 1% BSA Stain buffer, add 9µl of one phosph specific antibody/tube. Incubate 2 hours in dark at RT. Wash with 4mls FACS buffer, spin at 1000 RPM for 10 min at RT and re-suspend in 250µl FACS buffer. Flow Time! Day 1 B cell differentiation staining Aliquot 100,000 cells/FACS tube (about 100µl) Wash cells twice with a few ml of protein free PBS, spin at 1000 RPM for 10min at RT, resuspend in 1ml protein free PBS and add 1µl violet viability stain. Incubate 30min RT in dark. Wash cells once w/protein free PBS. Add antibodies (IgM-FITC, IgD-PE, B220-PerCP, CD19-PE-Cy7, CD43-APC) at 1:250 in PBS/1%FBS and incubate 20min RT in dark. Five stained, plus unstained control and seven individual stain controls is thirteen tubes total. Wash cells once w/ FACS buffer, spin at 1000 RPM for 10min at RT. Resuspend in 250µl FACS buffer. Flow Time! |
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