| This is a standard protocol used at Pharmingen for Quality Control testing of the anti-cyclin antibodies by flow cytometry. It is a good starting point. However, investigators may need to optimize protocols for their own experimental system. It is particularly useful to refer to the published literature regarding protocols typically used for a given type of protein. MATERIALS:
12 x 75 mm test tubes, Pipetman pipettes (P-20, P-200 and P-1000), 50 ml conical tubes, Pipet tips, Tabletop centrifuge or equivalent, Permanent marker, Aspirator BUFFERS:
Phosphate Buffered Saline (PBS): (Cat. No. 21428A; 3 bottles of 125 ml each): 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 dissolved in distilled, water. The pH has been adjusted to 7.2 using hydrochloric acid.
Wash Buffer: PBS/0.1% NaN 3 /1% heat-inactivated fetal bovine serum. The pH of the Wash buffer should be 7.1–7.4. REAGENTS: Wash buffer stored at 4°C, 75% ethanol stored at –20°C, Pure methanol stored at -20°C, 1% formaldehyde (methanol-free) in PBS, stored at 4°C, 0.25% Triton ® X-100 in Wash buffer, stored at 4°C, propidium iodide (PI) solution: 10 µg/ml PI in PBS, stored at 4°C. Procedure: | I. | Fixation | | | | 1. | Harvest, count and pellet cells following standard procedures. | | 2. | Wash cells once by resuspending the pellet in 30-40 ml of Wash buffer. Centrifuge at 200 x g for 10 min and aspirate supernatant. | | 3. | While vortexing, add 10 ml cold 75% ethanol (for D1, use pure cold methanol), drop by drop, to the cell pellet. | | 4. | Incubate at –20°C for a minimum of 2 hr. The fixed cells can be stored at –20°C in 75% ethanol for up to 30 days. Note: For D-type cyclins (besides D1) the cells should first be fixed in 5 ml of 1% methanol-free formaldehyde in PBS for 15 min on ice (or at 4°C) prior to fixation with ethanol. Following incubation with formaldehyde, centrifuge at 200 x g for 10 min and aspirate supernatant. Resuspend pellet in 30-40 ml Wash Buffer, centrifuge at 200 x g for 10 min and aspirate supernatant. Then go to Step No. 3 above. | | | II. | Staining | | | | 1. | Just prior to staining, remove ethanol by centrifugation at 200 x g for 10 min. Aspirate and wash once by resuspending pellet in 30-40 ml of Wash buffer. Centrifuge at 200 x g for 10 min. Aspirate supernatant. | | 2. | Add 5 ml cold 0.25% Triton ® X-100 in Wash buffer to the cell pellet, vortex and incubate 5 min on ice (or at 4°C). | | 3. | Add 30-40 ml Wash buffer to the above suspension and centrifuge at 200 x g for 10 min. Aspirate supernatant. | | 4. | Resuspend pellet in Wash buffer to a final concentration of 1 x 107 cells/ml. | | 5. | Aliquot 100 ml cell suspension (1 x 106 cells) into 12 x 75 mm tubes for staining. | | 6. | Add 20 ml of anti-cyclin or isotype control antibody at optimal working dilution. Incubate 30 min at RT in the dark. | | 7. | Add 2 ml Wash buffer, centrifuge for 5 min at 200 x g. Aspirate supernatant. | | 8. | If using directly conjugated isotype controls and mAbs, go to Step 10 below. | | 9. | If using unconjugated isotype controls and mAbs, add 20 ml of FITC-conjugated goat anti-mouse Ig (Cat. No. 12064D) at optimal working dilution. Incubate 30 min at RT in the dark. Add 2 ml Wash buffer, centrifuge 5 min at 200 x g Aspirate supernatant. | | 10. | Resuspend cell pellet in 0.5 ml PI solution for simultaneous analysis of DNA cell cycle and cyclin expression. Incubate cells at 4°C in PI for 20 min prior to analyzing by flow cytometry. Analyze stained cells within 4 hr. Store at 4°C in the dark prior to analysis. | | | |
