Intracellular cytokine staining

1.0  Purpose 
To define the procedure for Th1, Th2 and Tfh intracellular cytokine staining including two or more brilliant violet antibody conjugates.
 

2.0  Safety Considerations

   2.1 Individuals performing this procedure must have documented completion of Blood-borne Pathogen training and Working Safely with Nonhuman Primates training.

   2.2 Required Personal Protective Equipment: Closed front gowns, double gloves, face mask, safety glasses. Face mask and safety glasses can be replaced by a faceshield

   2.3 The procedure described is to be performed in a biosafety cabinet except the special considerations below.

         2.3.1 All centrifugation steps must be done using a rubber gasketed spill-proof centrifuge bucket and lid when working with un-fixed NHP samples

         2.3.2 Immediately following any centrifugation step, sample removal from the rubber gasketed spill proof container must be done in a Biosafety cabinet. 

3.0  Definitions

   3.1 ICS: Intracellular cytokine staining

   3.2 R10: RPMI 1640 with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 ug/ml streptomycin

   3.3 HINCS: Heat-inactivated newborn calf serum 

   3.4 DMSO: Dimethyl sulfoxide

   3.5 RT: Room temperature

   3.6 SEB - Staphylococcus Enterotoxin B 

4.0  Reagents and Materials

5.0  Preliminary Operations

   5.1 Determine desired plate layout.  One column per animal is recommended.  Each sample will likely have at least 2 stimulations each: peptide and negative control (DMSO only).  If staining more than 3 million cells per sample, cells should be plated in duplicate or triplicate.  If running SEB stimulation wells, skip several rows to avoid contamination.

   5.2 Prepare the peptide pool(s) by mixing the peptides in DMSO to achieve a final concentration of 400 μg/ml (prepare according to NIC-1002 SOP).  Peptides are used at a final concentration of 2 μg/ml when added to cells. Mark aliquots with a dot after each thaw. Do not thaw more than 3 times.

   5.3 Prepare SEB at a final concentration of 0.5 mg/ml in de-ionized water and store at -20^oC.  SEB is used at final concentration of 2 μg/ml when added to cells.
 

   5.4 In separate tubes, prepare the appropriate Stimulation Mixes (peptide and negative control) as listed below.  The table below is the amount of stimulation mix components PER WELL in 100ul.  Prepare enough solution for 14-15 wells for each full row (12 wells) needed.

       Note: NIC peptides are resuspended at 400 μg/ml while SEB is resuspended at 500 μg/ml. Therefore, use 1ml /test for peptides at 400 μg/ml (most SIV peptides) and 0.8 μl/ for SEB at 500 μg/ml.

   5.5 Determine the amount of Perm/Wash Buffer needed for the experiment and dilute the 10X Perm/Wash Buffer 1:10 with deionized water.  Shake well and filter through a 0.22 micron vacuum flip filter. Store at 4°C for 1 week.
 

   5.6 Prepare 25 ml of PBS + Benzonase per plate by diluting 5 μl of Benzonase nuclease stock in 25 ml of PBS.
 

   5.7 Prepare 1% formaldehyde by diluting the 4% stock formaldehyde solution in 1X PBS.  Make enough for 125 μl per sample (plus a little extra).
 

   5.8 Prepare the live/dead Amine Staining Mix no more than 1 - 2 hours prior to staining cells.  Dilute the appropriate amount of amine reactive dye 1:40 in deionized water, then 1:20 in 1X PBS at a total volume of 100 μl per sample (make a little extra).  Store at 4°C in the dark until ready to use.
 

   5.9 Prepare the Surface Staining Mix no more than 1 - 2 hours prior to staining cells.  Add the appropriate antibodies to appropriate Brilliant Stain Buffer in a microcentrifuge tube, 15 ml conical tube, or a 50 ml conical tube depending on the final volume (be sure to add the Brilliant Staining Buffer to the tube first!).  For volumes larger that 1.5 ml, split antibody mix evenly between microcentrifuge tubes and spin.  Spin the antibodies in a microcentrifuge at 13,000 rpm (15,700g) for 5 minutes to remove aggregates.  Remove the supernatant and adjust the total volume to 100 μl per sample (depending on panel) with Brilliant Staining Buffer. Store at 4°C in the dark until ready to use.

   5.10 Prepare the Intracellular Cytokine Staining Mix no more than 1-2 hours prior to staining cells.  Add the appropriate antibodies to BD Horizon Brilliant Staining Buffer in a microcentrifuge tube, 15 ml conical tube, or a 50 ml conical tube depending on the final volume (be sure to add the Brilliant Staining Buffer to the tube first!).  Spin the antibodies in a microcentrifuge at 13,000 rpm (15,700g) for 5 minutes to remove aggregates.  For volumes larger that 1.5 ml, split antibody mix evenly between microcentrifuge tubes and spin.  Remove the supernatant and add the appropriate volume of 10X Perm/Wash Buffer. Store at 4°C in the dark until ready to use.

6.0  Procedure

   6.1 Prepare cells according to the appropriate SOP (cell preparation and/or cell thawing SOP NIC-1004) using R10 as the final buffer.  Cells should be at a final concentration of 10 - 30 million cells/ml.
 

   6.2 Pipette 100 μl of cells into the appropriate wells on the plate (1 to 3 million cells/well) according to plate layout.

   6.3 Pipette 100 μl of the peptide and DMSO Stimulation Mixes from the table above into the appropriate wells and mix well with a multi-channel pipette.
 

   6.4 Incubate the plate for 6 hrs (18 hrs if overnight stimulation) in a humidified 5% CO₂ incubator at 37^oC.  At the end of the incubation, the plate may be held at 4^oC overnight if desired. Percival Run program: 14120901

   6.5 Centrifuge the plate at 2000 rpm (859g) for 3 minutes at RT (18-26°C).  All centrifugation steps from this point will be done the same way.

   6.6 Flick supernatant into a biohazardous waste container under a laminar flow hood and immediately and quickly blot the plate on a paper towel before returning to an upright position (see NIC-1007.1 ICS Appendix1.mov). Do not flick outside of the hood. All flicks and blots from this point will be done the same way.

   6.7 Add 200 μl of PBS + Benzonase to each of the wells and mix well by pipetting up and down with a multi-channel pipette.  Centrifuge, flick, and blot.
 

   6.8 Add 100 μl of the prepared Amine Staining mix to each of the wells and mix well.  Incubate for 20 min at RT protected from light.
 

   6.9 Wash the cells by adding 120 μl of Wash Buffer (4%HINCS in RPMI 1640 w/o phenol red) directly to the wells containing the cells in the Amine Staining Mix. Centrifuge, flick, and blot.

  6.10 Add 200 μl of Wash Buffer to the wells and mix well. Centrifuge, flick, and blot.

  6.11 Add 100 μl of Surface Staining Mix to each of the wells, mixing well.  Incubate for 20 min at RT protected from light.

  6.12 Wash the cells by adding 120 μl of Wash Buffer directly to the wells containing the cells in Surface Staining Mix. Centrifuge, flick, and blot.

  6.13 Add 200 μl of Wash Buffer to the wells and mix well.  Centrifuge, flick, and blot.

  6.14 Add 100 μl of Cytofix/Cytoperm reagent to each of the wells, mixing well. Incubate for 20 min at 4°C protected from light.

  6.15 Wash the cells by adding 120 μl of 1X Perm/Wash buffer directly to the wells containing cells in Cytofix/Cytoperm. Centrifuge, flick, and blot.

  6.16 Add 200 μl of 1X Perm/Wash buffer to the wells and mix well.  Centrifuge, flick, and blot.

  6.17 Add 100 μl of Intracellular Staining Mix to the wells, mixing well.  Incubate for 20 min at RT in the dark.

  6.18 Wash the cells by adding 120 μl of 1X Perm/Wash buffer directly to the wells containing cells in the Intracellular Staining Mix. Centrifuge, flick, and blot.

  6.19 Add 200 μl of 1X Perm/Wash buffer to the cells and mix well. Centrifuge, flick, and blot.

  6.20 Repeat step 6.19 for a third wash.

  6.21 Add 125 μl of 1X PBS to the cells and mix.

  6.22 Fix by adding 125 μl of 1% formaldehyde to the cells in 1X PBS for a total volume of 250 μl (0.5% formaldehyde).  Store covered in foil at 4°C until ready to run on the instrument (within 48 hours).

  6.23 Set up comp tubes within 1 – 2 hours of running samples on the instrument.

  6.24 Transfer cells to appropriate FACS tubes if not using the HTS.

7.0  Document History

Version 2:   

Added NOTE section to step 5.3. AN 7/9/19

Version 3:   

Removed costimulatory antibodies from step 5.3 and corresponding reagent information in 4.0. SA 3/9/21

Version 4:   

Added definitions and dilution information for SEB stimulation in 19-color panel in 3.6 and 5.4, respectively.  SA 7/20/21

Version 5: 

Changed the stimulation table calculations to PER WELL.  220408 SA 

Version 6: 1/9/23

Added 2.2 to specify required PPE. Added 2.3.1 and 2.3.2 to clarify the use of rubber gasketed centrifuge bucket lids and when they can be removed. DF