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P r o t o c o l s

Staining Procedure for Flow Cytometric Detection of Human Cyclins

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 7.17.4.

REAGENTS: Wash buffer stored at 4C, 75% ethanol stored at 20C, Pure methanol stored at -20C, 1% formaldehyde (methanol-free) in PBS, stored at 4C, 0.25% Triton X-100 in Wash buffer, stored at 4C, propidium iodide (PI) solution: 10 g/ml PI in PBS, stored at 4C.

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 20C for a minimum of 2 hr. The fixed cells can be stored at 20C 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 4C) 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 4C).

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 4C in PI for 20 min prior to analyzing by flow cytometry. Analyze stained cells within 4 hr. Store at 4C in the dark prior to analysis.

Protocols Index>>
Flow Cytometry
Fluorochrome Absorption and Emission Spectra
Procedure for Setting Compensation for Multi-Color Flow Cytometric Analysis
Cell Fixation/Permeabilization Kits
Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis
Annexin V Staining Protocol
APO-BRDU™ Procedure
APO-DIRECT™ Procedure
Detection of BrdU Incorporation in DNA Synthesizing Cells
Staining Procedure for Flow Cytometric Detection of Human Cyclins
Indirect Immunofluorescence Staining of Human Platelets
Immunofluorescence Staining of Human Cells by Lysed Whole Blood Method
Immunofluorescent Staining of Mouse and Rat Leukocytes
The Uses of Fc Block in Immunophenotyping of Mouse or Rat Leukocytes
ELISA/ELISPOT
Cytokine ELISA
Mouse IgE ELISA Protocol
Immunohistochemistry
Preparation and Staining of Frozen Tissue Sections
Preparation and Staining of Paraffin Sections
Microwave citrate Pretreatment of Paraffin Sections
Immunohistochemistry / Tissue Section Staining
Cell Staining for Immunofluorescence Microscopy
Western Blotting
Western Blotting with Alkaline Phosphatase Conjugates
Western Blotting with Biotinylated Antibodies
Western Blotting with Horseradish Peroxidase Conjugates
Western Blotting with Monoclonal Antibodies
Western Blotting with Rabbit Polyclonal Antibodies
Preparation of Brain Membrane Fractions for Western Blot Analysis
Immunoprecipitation
Immunopurification of Tyrosine Phosphorylated Proteins
Immunoprecipitation with Antibody:Agarose Conjugates
Immunoprecipitation with anti-Phosphotyrosine: Biotin Conjugates
Immunoprecipitation With Soluble Antibodies
Immunoprecipitation of 35S Labeled Cells
RNAse Protection Assay (RPA)
RNase Protection Assay
Total RNA Isolation
Protein Kinase Assay (PKA)
Protein Kinase Assay with an Immunocomplex
Protein Kinase Assay after Denaturation and Renaturation




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