General Procedure for Intra-Cellular Staining

  1. (a) Suspension cells: Transfer cells to a 15 ml conical tube and centrifuge at 1000-1200 rpm for 5 minutes. Discard media and wash cells 2X with 5 ml 1X dPBS. (b) Adherent cells: Trypsinize cells with Trypsin/0.25% EDTA. neutralize the trypsin with an equal volume of media once the cells are detached. Transfer cells to a 15 ml conical tube and centrifuge at 1000-1200 rpm for 5 minutes. Discard media and wash cells 2X with 5 ml 1X dPBS.
  2. Re-suspend cells in 1 ml CytoFix/CytoPerm Buffer (BD Biosciences Catalog No. 554722). Incubate for 30 minutes at 4°C in the dark.
  3. Wash cells 2X in 5 ml 1X BD Perm/Wash buffer (BD Biosciences Catalog No. 554723). The Concentrated 10X Perm/Wash buffer must be diluted in dPBS to 1X. All centrifugation steps must be performed for 5 minutes at 1000-1200 rpm.
  4. Re-suspend cells in 10% normal human serum to block Fc receptors and subsequently reduce non-specific binding. The normal human serum should be free of endogenous IFN of interest. Incubate for 20 minutes at room temperature (RT).
  5. Wash cells 1X in 5 ml 1X BD Perm/Wash buffer. The centrifugation must be performed for 5 minutes at 1000-1200 rpm.
  6. Re-suspend cells in a volume of 1X BD Perm/Wash buffer such that there is sufficient volume for each sample (100 μl of cell volume is recommended per sample). Note: At least 100,000 cells are required per sample.
  7. Distribute the cells into tubes with one tube per sample. We recommend using 5 ml polystyrene round bottom tubes (BD Falcon Catalog No. 352054).
  8. Add labeled antibody from PBL InterferonSource to the samples. It is recommended to try different concentrations of the labeled antibody to optimize for the correct staining concentration in the assay system. We recommend stain one set of negative control cells with labeled Mouse IgG isotype control at the same concentration as that of the labeled antibody. If several staining concentrations of the labeled antibody are tested, dilute the isotype control to a concentration that falls in the middle of the range.
  9. Vortex the tubes.
  10. Incubate for 30 minutes at room temperature in the dark.
  11. Wash cells 2X with 0.5-1 ml of 1X BD Perm/Wash buffer (BD Biosciences catalog No. 554723). All centrifugation steps must be performed for 5 minutes at 1000-1200 rpm. Vortex the tubes between each re-suspension in 1X BD Perm/Wash buffer.
  12. Re-suspend cells in 0.5 ml 0.5% paraformaldehyde if the acquisition will be done 12-24 hours after the staining process. The cells can be re-suspended in 0.5 ml dPBS if the acquisition will be done immediately.

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