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Department of Microbiology and Immunology: Flow Cytometry Core Facility

Immunofluorescence Staining for Immunotyping

Surface Staining

  1. Aliquot the appropriate amount of cells into a 96 well round-bottom plate or into FACS tubes whatever is preferred method. Spin at 1500rpm for 2-3 minutes (use this speed and time for all spins).
  2. Dump off media and wash in FACs buffer. Spin cells to pellet.
  3. Dump off FACS buffer. Add 50ml of Fc block (100ml 24G.2 Ab, 5ml normal rat IgG and 895ml FACS buffer/ml). Incubate cells at 4°C for 10-15 minutes while you prepare the surface stain Ab cocktail.
    1. Additionally, if you are using a live/dead stain dilute the live/dead stain in Fc block (1:1000) and add 50ml per well and incubate at 4°C for 10-15 minutes.
  4. Dilute the staining panel of antibodies in 50ml of FACS buffer. Final volume for staining is 100ml; so take this into account when diluting the antibodies.
  5. Stain cells for 20-30 minutes at 4°C covered in foil.
  6. Wash cells 2-3 times in FACS buffer. If running cells right after staining there is no need to fix the cells. If the cells will not be run immediately fix in 4% paraformaldehyde (50ml/well) for 10 minutes at room temperature covered in foil.
  7. Wash cells after fixation and resuspend in 200ul for flow cytometry. Pass cells through a 40mm cell strainer-topped test tube prior to running on the cytometers. Store cells covered in foil at 4°C until ready to run.

Annexin V Staining

  1. Follow the protocol for surface staining.
  2. Wash cells in FACS buffer and then in 1 x binding buffer supplied with the BD Annexin V kit.
  3. Add 2.5ml Annexin V and 10ml Propidium iodide/100ml 1 x binding buffer/well. Stain 15 minutes at room temperature covered in foil.
  4. Wash 2-3 times in 1 x binding buffer.
  5. Resuspend in FACS buffer (200ml) and run samples within 1 hr of staining.

Intracellular Staining of T Cells

  1. Follow protocol for surface staining. Fix cells in 4% paraformaldehyde for 10 minutes.
  2. Following fixation permeabilize the cells by adding 100ml of Perm buffer (0.1% saponin in FACS buffer) to each well. Spin immediately.
  3. Make up the Ab cocktail in the perm buffer and add 100ml/well.
  4. Stain cells for 20-30 minutes at 4°C covered in foil.
  5. Wash cells twice in perm buffer and once in FACS buffer.
  6. Resuspend the cells in 200ml of FACS buffer and filter samples prior to running.

Intracellular staining of Tbet, Bcl6, Foxp3, KI-67, Bcl2, etc. in T cells

  1. Follow the protocol for surface staining.
  2. After washing off the surface stain use the eBioscience Foxp3 kit for fixing and staining.
  3. Dilute the fix/perm buffer 1:3 in dilutant. Add 100ml/well and fix 30 minutes at 4°
  4. Wash with 1x perm/wash buffer (dilute 10x stock with water). Can leave cells overnight in perm/wash buffer.
  5. Make up Ab panel cocktail in perm/wash buffer. Stain in 100ml volume for 20-30 minutes at 4°C covered in foil.
  6. Wash twice in perm/wash buffer and one time in FACS buffer.
  7. Resuspend in FACS buffer (200ml) and filter before running.

Intracellular Staining for BrdU

  1. Follow the protocol for surface staining. After washing use the BD BrdU kit for staining for intracellular BrdU.
  2. Fix cell in 50ml in 1 x BD Fix/Perm buffer for 30 minutes at room temperature covered in foil.
  3. Wash with 1 x BD Perm/Wash buffer.
  4. Incubate cells in 25ml cytofix/cytoperm buffer for 10 minutes on ice, covered.
  5. Wash with 1 x BD Perm/Wash buffer.
  6. Fix again in 50ml 1 x Fix/Perm buffer for 5 minutes on ice, covered.
  7. Wash with 1 x BD Perm/Wash buffer.
  8. Resuspend cells in 300ug/ml DNAse in 1 x PBS (100ml/well) and incubate for 1 hr at 37°
  9. Wash with 1 x BD Perm/Wash buffer.
  10. Stain for BrdU (1:25) diluted in 1 x BD Perm/Wash buffer (50ml/well) and incubate at room temperature for 20 minutes.
    1. If co-staining for other intracellular proteins stain in the same volume, but do so at 4°
  11. Wash three times with 1 x BD Perm/Wash buffer.
  12. Resuspend in 200ml of FACS buffer and filter before running.

Quenching Anti-Human Fc Ab

  1. If staining with an antibody conjugated to human Fc you will need to add a secondary step to come in with the anti-human Fc antibody conjugated to the appropriate fluorochrome.
  2. First to prevent high background staining the anti-human Fc Ab must be incubated with Fc block made up in the appropriate perm buffer for 30 minutes on ice.
  3. Following absorption the anti-human Fc antibody can be added to the cells for staining at 4°C covered in foil. Stain for 20-30 minutes.

SA Staining

  1. If a biotinylated Ab is used in any of the staining protocols then a secondary staining step must be added following the initial stain with the biotinylated Ab.
  2. Dilute the SA-fluorochrome-labeled Ab in the appropriate buffer and stain for 5 minutes at 4°C covered in foil.
  3. Wash three times before resuspending in 200ml of FACS buffer.

FACS buffer (1L)                              Macrophage FACS buffer (1L)

2g        BSA                                         5g        BSA

2ml      0.5mM EDTA                         2ml      0.5mM EDTA

.1%      sodium azide                          .1%      sodium azide

1 x PBS to 1L                                      1 x PBS to 1L

LYSING

Use 0.86% NH4Cl made in sterile autoclaved water to lyse our RBCs. We use 10 ml per spleen and incubate for 10 minutes. We neutralize the lysis buffer with 1x PBS (30 ml) and then spin for 10 minutes at 1200 rpm before counting