FACS - Methods

Methodologies used for Flow Cytometry

Below are several methods commonly used in flow cytometry for cell staining. Some are for surface staining, cytoplasmic staining, DNA staining or DNA/cytoplasmic staining. In many cases the staining method needs to be adapted to the particular cells of interest and the staining conditions optimized for your particular system. It is recommend that you consult with Karla Gorena prior to your first experiment so that I can suggest possible modifications to your staining procedures in order to save you time, money, and possibly re-doing your initial experiment(s).


EXAMPLES FOR IMMUNOFLUORESCENCE STAINING

I. Dual color direct staining of intact blood cells: example; CD34+/CD45- hematopoietic stem cells
Staining is performed by two-color immunofluorescence for the CD34/CD45- antigens.
  • 1. Tubes containing 2x106 nucleated cells are prepared and red blood cells (RBCs) are lysed by a commercial lysing reagent [Becton Dickinson (B.D.), San Jose, CA as per manufacturer's instructions].
  • 2. Alternatively, RBCs could be depleted by Ficoll-Hypaque gradient centrifugation.
  • 3. Twenty ul of anti-CD34 (HPCA-2-PE) and 20ul of anti CD45-FITC, or 20 ul of IgG1/IgG2 Simultest Control (both from B.D) are aeddd to a 100ul of cell suspension (1x107 cells/ml) and tubes incubated for 30 min. at 4°C.
  • 4. Unbound antibodies are washed by centrifugation (at 250xg for 5 min.) and pellet resuspended in 0.5ml of PBS buffer.
  • 5. Tubes should be analyzed within 24 hours.
  • 6. For later time analysis, cells at step 4 should be fixed by adding to the cell pellet 0.5ml of 2% formalin and storing at 4°C protected from light. For best results, the tubes should be analyzed within 3 days.
  • 7. Results should be corrected for background staining by subtracting the value obtained from the IgG1/IgG2--Simultest Control tubes.
  • COMMENTS: Protect tubes from light at all times! For staining of cells from tissue culture, omit step 1 and2. For single color add 1 antibody and 1 corresponding Ig control. For indirect staining (single color) perform step 1-2 and add primary (unlabeled) antibody in step 3. Perform step 3 and 4 and then add secondary (labeled) antibody (e.g. anti mouse, anti rat, anti rabbit, anti goat for primary mouse, rat, rabbit or goat antibodies, respectively). Repeat step 3 and 4.

II. DIRECT STAINING FOR CYTOPLASMIC CD3 AND NUCLEAR TdT (fixed cells)
Reagents:
  • 1. PBS + 2% B.S.A.
  • 2. PermeaFix---to fix and permeate cells
  • 3. Anti CD 3 Antibody (PE), DAKO
  • 4. Anti TdT Antibody (FITC), Immunotech
  • 5. IgG1 (FITC) IgG2 (PE), DAKO
Procedure:
  • 1. Label 2 tube for this staining
  • 2. Count cells from flask and remove the 1 X 106 cells for each tube needed.
  • 3. Wash cells (x2) with PBS + 2% BSA centrifuge for 5 minutes at 1000 rpm at room temperature.
  • 4. Remove all but 50 ml of the supernatant by aspiration.
  • 5. Add 0.5 ml of PermeaFix and mix.
  • 6. Incubate tubes at room temperature for 40 minutes.
  • 7. Centrifuge for 5 minutes at 1400 rpm (=250g) at room temperature.
  • 8. Remove all but 50 ml of the supernatant by aspiration.
  • 9. Wash cells (x3) with PBS + 2% BSA Remove all but 50 ml of the supernatant by aspiration.
  • 10. Re suspend the pellets in 2 ml of PBS + 2% BSA and incubate for 10 minute room temperature.
  • 11. Centrifuge for 5 minutes at 1400 rpm at room temperature.
  • 12. Remove all but 50 ml of the supernatant by aspiration.
  • 13. Add 7.5 ml of IgG 1 & IgG 2a to the bottom of tube marked 1.
  • 14. Add 7.5 ml of anti CD3 antibody and 10 ml of anti TdT antibody to the bottom of tube marked 2.
  • 15. Mix and incubate all tubes at 4°C temperature for 45 minutes.
  • 16. Wash cells times two with PBS + 2% BSA by centrifugation for 5 minutes at 1400 rpm at room temperature.
    COMMENTS: Protect tubes from light at all times! If using blood, deplete red blood cells first.

III. STAINING WITH PROPIDIUM IODIDE (PI) FOR CELL CYCLE ANALYSIS OF UNFIXED CELLS
  • 1. COLLECT 0.5-1.0 MILLION CELLS/TUBE AND SPIN AT 1,400 RPM (250g) x5min. REMOVE SUPERNATANT AND LEAVE "DRY"PELLET.
  • 2. TO THE PELLET ADD 1 ML OF P.I. HYPOTONIC BUFFER.
    (10mM TRIS-HCl BUFFER, pH 7.4 PLUS 5mM MgCl2)
  • 3. MIX WELL.
  • 4. ADD 30 uL OF P.I. STAINING BUFFER (STOCK of 1 mg/ml OF P.I. PLUS 100ug/ml OF RNAse A),
    BOTH FROM SIGMA, IN HYPOTONIC BUFFER). Mix.
  • 5. FINAL CONCENTRACTION OF P.I. IS 10 ug/ml IN CELL SUSPENSION AND RNAse A IS 1 ug/ml.
  • 6. MIX WELL AND INCUBATE ON ICE FOR 1 HOUR COVERED.
  • 7. KEEP AT 4°C FOR >3 HOURS AND ANALYZE.
  • COMMENTS: Tubes are stable for 2-3 days, at 4�C, if protected from light. This method works best with lymphoid cells and less with adherent cells (e.g., epithelial or fibroblast type of cells). This method preserves membrane/nuclear staining.
    Reference: Gazitt, Y., Deitch, A.D., Marks, P.A., and Rifkind, R.A. Cell volume changes in relation to the cell cycle in differentiating and non-differentiating murine erythroleukemia cell. Exp. Cell. Res., 117:413-420, 1978.

IV. STAINING WITH PROPIDIUM IODIDE (PI) FOR CELL CYCLE ANALYSIS OF FIXED CELLS
  • 1. COLLECT 0.5-1.0 MILLION CELLS/TUBE AND SPIN AT 1,400 RPM (250g) x5min. REMOVE SUPERNATANT AND LEAVE "DRY"PELLET.
  • 2. TO THE PELLET ADD 1 ML OF 70% ICE COLD METHANOL. (keep in -20°C)
  • 3. MIX WELL. Incubate for an hour on ice. Spin at 450gx5min. Remove supernatant by suction. Wash (x2) with 1 ml PBS each time by spinning at (400gx5min.) with PBS.
  • 4. TO THE FINAL PELLET ADD! ML OF PBS PLUS uL OF P.I. STAINING BUFFER (STOCK of 1 mg/ml OF P.I. PLUS 100ug/ml OF RNAse A), BOTH FROM SIGMA, IN PBS. Mix well.
  • 5. FINAL CONCENTRACTION OF P.I. IS 10 ug/ml IN CELL SUSPENSION AND RNAse A IS 1 ug/ml.
  • 6. MIX WELL AND INCUBATE ON ICE FOR 1 HOUR COVERED.
  • 7. KEEP AT 4°C FOR >3 HOURS AND ANALYZE.
  • COMMENTS: Tubes are stable for 1-2 days, at 4�C, if protected from light. This method works with most cell types, however some nuclear and cytoplasmic antigens are destroyed. So this method does not always work for dual/triple staining.

V. Propidium Iodide Staining Procedure for DNA analysis of fixed cells
Cell Fixation:

Obtain a bucket of ice, place 70% ethanol at -20°C (1ml/sample), and turn on refrigerated centrifuge.

  • 1. Wash cells from plate by pipetting up and down vigorously until cleared.
  • 2. Centrifuge for 10 min, at 1000 RPM, 4oC, then aspirate.
  • 3. Add 5 ml PBS (4oC).
  • 4. Centrifuge for 10 min, at 1000 RPM, 4oC, then aspirate. 9:43
  • 5. Add 5 ml PBS (4oC).
  • 6. Centrifuge for 10 min, at 1000 RPM, 4oC, then aspirate.
  • 7. Add 1ml PBS (4oC).
  • 8. Remove 8ml (x2) for cell counting. Note total cell number remaining.
  • 9. Centrifuge the remaining 984ml for 10 min, at 1000 RPM, 4oC, then aspirate.
  • 10. Vigorously vortex pellet for 10 sec.
  • 11. Continue to vortex, while adding 1ml 70% ethanol drop wise. Fix in 70% ethanol overnight at 4oC. If storing for longer periods, place at -20oC.

Staining:

Prepare 1mg/ml RNase A (100ml for each sample). Must be made Fresh. (Use 1 to 2 million cells per sample in B-D Falcon #2052 or #2054 12x75mm (5ml) tubes.)
  • 1. Briefly vortex.
  • 2. Centrifuge for 5min, at 2800 RPM, 4oC, then aspirate.
  • 3. Gently vortex.
  • 4. Add staining solution: 800ml PBS, 100ml of 0.4mg/ml Propidium Iodide soln. (final [40mg/ml]), 100ml of 1mg/ml RNaseA (final [0.1mg/ml]).
  • 5. Incubate at 37oC for 30 min.
  • 6. Use a 1cc syringe to pass cell solution through the cell sifter or nylon mesh.
  • 7. Leave on ice or in the refrigerator until ready to analyze.
References:
Michael G. Ormerod. 1998. Flow Cytometry of Apoptotic Cells. Cell Biology: A Laboratory Handbook. 1:351-356. Philip D. Noguchi. 1991. Use of Flow Cytometry for DNA Analysis. Current Protocols in Immunology. 5.7.1-5.7.6.

VI. Processing and Staining of Cells for FACS DNA analysis for adherent fixed cells (ethanol/Triton X-100 method)
  • 1. Wash the cells with 5 ml PBS
  • 2. Trypsinize cells with 2 ml Trypsin-EDTA
  • 3. Add 2 ml PBS and resuspend the cells
  • 4. Pellet the cells at 5000rpm in clinical centrifuge at RT for 3 to 5 minutes
  • 5. Aspirate and resuspend the cells in 2ml 70% ethanol and keep at RT for 20 minutes
  • 6. Store the cells at 4oC (if not processing for Propidium Iodide (PI) staining immediately)
  • 7. Pellet the cells at 5000rpm in clinical centrifuge at RT for 3 to 5 minutes
  • 8. Aspirate and resuspend the cells in 3 to 5 ml of PBS
  • 9. Pellet the cells at 5000rpm in clinical centrifuge at RT for 3 to 5 minutes
  • 10. Aspirate and resuspend the cells in 5 ml of DNA staining solution and keep at RT for about an hour in dark. DNA Staining Solution
    For 10 ml: 50 ul -Triton X 100:
    • 1 ml -10X PI (500 ug/ml)
    • 200 ul - RNase A (25 mg/ml)
    • 8.75 ml - PBS
  • 11. Filter the cells through Nylon cloth in to glass tubes (Falcon-352054)
  • 12. Give the cells for FACS Analysis (Keep the cells at 4oC if not given for FACS Analysis right away)
COMMENTS Since Triton X-100 is involved in this staining procedure cytoplasmic as well as membrane proteins may be lost.