FACS Protocol: Surface and Intracellular Staining of Human Whole Blood

This set of protocols is used for both basic surface staining of human (and monkey) whole blood, as well as staining for intracellular antigens such as perforin, Ki67, Bcl2, granzyme B, etc. For intracellular staining, it uses the BD Permeabilization Solution, which irreversibly forms holes in the cell membrane, and does not require the continued presence of detergent during the staining and early washing steps. Alternative protocols use saponin detergent (e.g. the BD Cytofix/Cytoperm kit; PDF of TDS), which reversibly forms holes in cell membranes, and which must be included throught the portions of the protocol in which intracellular antigens are targeted. We tend to use the reagents from BD and Pharmingen, but other suppliers have excellent equivalents.

Materials and Equipment

Reagents

Antibodies

Equipment

Cells

Preparation

Procedure

Surface Staining of whole blood

  1. Add antibodies and tetramers at appropriate volumes/concentrations to FACS tubes.

    As for all FACS staining, take reasonable measures to minimize exposure of fluorochrome antibodies to light; heroic measures are not needed.

    Note: Unless otherwise indicated, all antibodies are used at 10 µl (per 100 µl blood in step 2; adjust by an appropriate factor if more or less blood is used). The exception for surface-stains is HLA-DR PE (2 µl).

    Note: All tetramers are used at a concentration of 1:300 relative to the Altman Lab master stocks; for PE, these are approximately 0.5 mg/ml in MHC, and for APC, these are approximately 0.73 mg/ml in MHC.

  2. Aliquot 100 µll whole blood to each tube, vortex on high (setting: 8) for several seconds and incubate at room temperature for exactly 10 min. Incubation should be in the dark.
  3. Add 2 ml FACSlyse to each tube, vortex (setting ~5.5: until funnel forms) and incubate at room temperature for 10 min. exactly.
  4. Centrifuge at1600 rpm for 5 minutes at ~20 °C.
  5. Vacuum-pipette the supernatant and resuspend the cells in the remaining supernatant (~200 µl) by vortexing on high for at least 5 seconds (setting: 8).
  6. Add 3 ml staining buffer to each tube, vortex gently (setting ~4.5: until funnel forms) and centrifuge at 1600rpm for 5 minutes at ~20 °C.
  7. Repeat steps 5-6 once.
  8. If you are performing intracellular staining, proceed to step 1 below. Otherwise, disrupt the cell pellet by vortexing for at least 5 seconds, and add 200-300 µl of 1% PFA to the cells. Vortex again for 5 seconds. Acquire immediately, or store at 4 °C in the dark for acquisition within 24 horus.

Intracellular staining

  1. Vacuum-pipette the supernatant and resuspend the cells in the remaining supernatant by vortexing on high for at least 5 seconds (setting: 8).
  2. Add 500 µl FACSperm to each sample and incubate at room temperature (~25 °C) for exactly 30 minutes. Incubation should be in the dark.
  3. Add 3 ml staining buffer to each tube, gently vortex (setting: ~4.5: until funnel forms) and centrifuge at 1600rpm for 5 minutes at ~25 °C.
  4. Vacuum-pipette the supernatant, leaving ~200 °l covering the cells, and resuspend the cells by vortexing on high for at least 5 seconds (setting: 8).
  5. Add intracellular antibodies at appropriate concentrations and incubate at room temperature (~25 °C) for 30 minutes.  Incubation must be in the dark.

    Note: Unless otherwise indicated, all intracellular antibodies are used at 10 µl (per 100 µl of blood).  The exception is Granzyme B PE (0.75 µl)

  6. Repeat steps 3 and 4 twice.
  7. Add 200-300 µl paraformaldehyde 1% to each sample. Vortex for at least 5 seconds. Immediately acquire data on the flow cytometer, or store the samples at 4 °C for up to 24 hours before acquisition.

Flow