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This is a standard protocol used at Pharmingen for Quality Control testing
of the anti-cyclin antibodies by flow cytometry. 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. 554781; 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% NaN3 /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 detection
of cyclin 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 ml 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 µl 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 µl 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 µl
of FITC-conjugated goat anti-mouse Ig (Cat. No. 554001) 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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