Cytokine release assay

Flow cytometry analysis of the CAR-transduced cells

Samples were run on the Gallios Flow Cytometer (Beckman Coulter), BD Accuri C6 Flow

Cytometer (BD) or the BD LSRII (BD).

1. Transfer 1-5x10⁵ cells per tube
2. Wash cells with 1-3 mL FACS buffer (1xPBS with 1% FBS or goat serum and 0.01% sodium azide)
3. Centrifuge the tubes at 400xg for 5 minutes and aspirate supernatant or decant (Leave about 150μL -200 μL supernatant)
4. For testing pancreatic cancer cell lines, use HER2PE and IgG PE isotype control. Add 20 ul of HER2PE (BD, 340552) or 2 ul of IgG1PE isotype (BD, 340761) to appropriate tubes, mix well by gentle vortex. Incubate for 30 min at 4C in dark covered with aluminum foil, wash twice with FACS buffer as described in steps 2 and 3 and run the samples on flow cytometer.
5. For HER2CAR transduced T cells, HER2 staining was performed by either
   1. using HER2-Fc chimera protein (3 μl at 0.1 μg/μl; R&D Systems, 1129-ER-050) and goat anti-human IgG1 Fc-PE (0.5 μl; eBioscience, 12-4998-82). or more recently tested
   2. using Goat anti-mouse IgG F(ab)2 conjugated to AF647: Jackson ImmunoResearch Cat# 115-605-006 and Goat IgG-AF647 (Isotype): Jackson ImmunoResearch Cat# 005-600-003
6. For HER2Fc-chimera staining, incubate cells with the recombinant HER2-Fc chimera (3μL at 0.1 ug/ul) protein for 30 min to 60 min at 4C in dark covered with aluminum foil, wash twice with FACS buffer and then stain for another 30 min with IgG1 Fc-PE (0.5μL) at 4C in dark covered with aluminum foil, wash twice with FACS buffer.
7. For IgG F(ab')₂ staining, add 1 μL of IgG F(ab')₂ specific and 1 μL of IgG into appropriate tubes(s) and mix well by gentle vortex, incubate for 30 min to 60 min at 4C in dark covered with aluminum foil, wash twice with FACS buffer.
8. Following HER2 staining, perform surface staining for other markers using anti-human CD161-APC (5 μl; BioLegend, 339912, Clone HP-3G10), CD3–fluorescein isothiocyanate (20 μl; BD Biosciences, 56180), or CD8-phytoplankton (PerCP) (20 μl; BD Biosciences, 347314) by incubating the samples with the antibodies for 30 min at 4C in dark covered with aluminum foil.
9. If surface stain only: Wash samples with FACS buffer twice, centrifuge at 400xg for 5 minutes and aspirate or decant the supernatant (Leave about 150μL -200μL supernatant) and proceed to Step 26. If staining for intracellular markers, proceed to Step 10.
10. After surface stain, add 1 mL FACS and spin down at 400xG for 5 min @ 4C.
11. Flick off supernatant, thoroughly resuspend cells in 1 mL FACS by vortexing. Spin down at 400xG for 5 min @ 4C.
12. Repeat Step 11.
13. While cells are spinning, make required volume of eBioscience FoxP3 FixPerm buffer by mixing 4X FixPerm concentrate with 1X FixPerm diluent in 1:3 ratio. 
14. After second full wash, flick off supernatant and thoroughly resuspend cells in 200 uL FixPerm buffer by vortexing. Incubate for 1 hour at 4C.
15. During incubation, make required volume of eBioscience FoxP3 Permeabilization buffer (“Perm buffer”) by mixing 10X Permeabilization buffer concentrate with ddH₂O in a 1:9 ratio.
16. After 1 hour FixPerm incubation, spin cells down at 400xG for 5 min @ 4C. Cell pellets from this stage on will be more transparent and harder to visualize.
17. Flick off supernatant, thoroughly resuspend cells in 1 mL Perm buffer by vortexing. Spin down at 400xG for 5 min @ 4C.
18. Repeat Step 17.
19. While cells are spinning, make required volume of intracellular antibody cocktail, using Perm buffer as the staining buffer. Commonly used titration for intracellular Abs: 1:50.
20. After second full perm wash, flick off supernatant and thoroughly resuspend cells in 100 uL intracellular antibody cocktail. (prepared in Step 19) by vortexing. Incubate for 1 hour at 4C.
21. After 1 hour intracellular stain incubation, add 1 mL Perm buffer to each sample and spin cells down at 400xG for 5 min @ 4C.
22. Flick off supernatant, thoroughly resuspend cells in 1 mL Perm buffer by vortexing. Spin down at 400xG for 5 min @ 4C.
23. Repeat Step 22.
24. After second full perm wash (Step 14), flick off supernatant and thoroughly resuspend cells in 1 mL FACS buffer by vortexing. Spin down at 400xG for 5 min @ 4C.
25. Flick off supernatant and thoroughly resuspend cells in 200 uL FACS buffer by vortexing.
26. Run samples on flow cytometer.
27. For single color controls, and proper compensation, stain VersaComp antibody capture beads with the same set of antibodies following the protocol of T.Byrd et al https://doi.org/10.1002/cyto.a.22717.
28. Briefly, each bottle of PSMS (VersaComp Antibody capture beads, Beckman Coulter, Brea, CA) contains ∼10 × 10⁶ PSMS/ml. For single color controls, add one 50 μl drop (5 × 10⁵ ) of coated PSMS and one 50 μl drop (5 × 10⁵ ) of uncoated PSMS for a total of 1 × 10⁶ PSMS per tube.
29. Stain each tube per manufacturer's recommendation for ≤1 × 10⁶cells with a different antibody from the T cell panel for 20 min at room temperature in the dark. Wash tubes containing PSMS in two milliliters of phosphate buffered saline (PBS; Sigma-Aldrich, St Louis, MO) containing 1% fetal calf serum (FCS; HyClone™, Thermo Scientific, Logan, UT) and centrifuge at 400g for 5 min.
30. Decant excess buffer leaving the PSMS pellet in a residual volume of 100 μl. Briefly vortex the tubes and then assay for expression of cell surface marker antibodies.
31. Alternatively, T cells were also stained with individual antibodies separately for single color and compensation controls at the same concentrations and conditions as described for transduced cells. 
32. Data analysis was conducted on ≥10,000 events with FlowJo version 10.0.00003 (Tree Star Inc.) for iOS (iMac Operating System).