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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 Na2
HPO4
, 1.8 mM KH2
PO4
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:
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I.
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Fixation
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1.
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Harvest, count and pellet cells following standard procedures.
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2.
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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.
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3.
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While vortexing, add 10 ml cold 75% ethanol (for D1, use pure cold methanol), drop by drop, to the cell pellet.
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4.
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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.
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II.
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Staining
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1.
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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 m
l of Wash buffer. Centrifuge at 200 x g for 10 min. Aspirate supernatant.
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2.
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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).
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3.
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Add 30-40 ml Wash buffer to the above suspension and centrifuge at 200 x g for 10 min. Aspirate supernatant.
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4.
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Resuspend pellet in Wash buffer to a final concentration of 1 x 107
cells/ml.
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5.
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Aliquot 100 m
l cell suspension (1 x 106
cells) into 12 x 75 mm tubes for staining.
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6.
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Add 20 m
l of anti-cyclin or isotype control antibody at optimal working dilution. Incubate 30 min at RT in the dark.
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7.
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Add 2 ml Wash buffer, centrifuge for 5 min at 200 x g. Aspirate supernatant.
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8.
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If using directly conjugated isotype controls and mAbs, go to Step 10 below.
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9.
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If using unconjugated isotype controls and mAbs, add 20 m
l 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.
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10.
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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.
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