β mercaptoethanol

For cell lines, ImProB^{15 (link)}, A-20^{31 (link)}, Ramos, and i.29 IgA^{32 (link)} electroporations were performed with 18.3pmol Cas9 and 22pmol gRNA, at 1.0E5cells/μl in OptiMEM (31985-047, gibco) using 10 μl tips in a Neon electroporation system (Invitrogen). For the human cell line, parameters were: 1350 v 30 ms 1 pulse and for the mouse cell lines: 1600v 20 ms, 1 pulse. All cell lines were grown in 1640 RPMI (01-100-1A, Biological Industries) supplemented with 10% HI FBS (04-127-1A, Biological Industries), 50 μM β-Mercaptoethanol and P/S (03-031-1B, Biological Industries). Transductions were performed with a 50,000 MOI of rAAV-DJ for mouse cell lines, and a 130,000 MOI of rAAV-DJ for human cell line. Efficiency of editing was determined 3 days following electroporation.
For plasmid electroporations we used 3 µg plasmid DNA/1E06 cells/10 µl Neon tip.
The Ramos cell line (ATCC CRL-1596) was provided by the Benhar lab, Tel Aviv University. The i.29 cell line was provided by the Zan Bar lab, Tel Aviv University. The ImProB cell line was provided by the Deriano lab, Pasteur Institute. The A-20 cell line was provided commercially (ATCC TIB-208).

B16 cells-expressing ovalbumin (courtesy of Lea Eisenach, Weizmann institute of Science) were suspended in DMEM medium w/o phenol red, supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, 100 mg/ml of streptomycin, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 50 mM β-mercaptoethanol (Biological Industries). Cells were seeded 50–100 × 10³ B16 cells per well in a 24-well plate; 150–300 × 10³ OT-I T-cells pre-activated and cultured for 7 days, were added on top of the B16 cells. After 24–48 h, cells were collected, and a single-cell suspension was prepared and stained with live/dead stain (L23105, Life Technologies, Carlsbad, CA, USA), CD8 (BLG100734, Biolegend), FasL (BLG106606, Biolegend), and PD-1 (BLG135225, Biolegend). Cells were then fixated (BLG420801, Biolegend), permeablized (BLG521002, Biolegend), and stained for intracellular granzyme B (BLG515406, BioLegend). The mean fluorescent intensity of Granzyme B, FasL, and PD-1 of live CD8⁺ cells was measured and analyzed using flow cytometry (Becton Dickinson; FlowJo software).

B16 cells expressing ovalbumin coupled with GFP (courtesy of Guy Shakhar, Weizmann institute of Science) were suspended in RPMI 1640 medium w/o phenol red, supplemented with 10% serum, 100 U/ml of penicillin, 100 mg/ml of streptomycin, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 50 mM β-mercaptoethanol (Biological Industries). Cells were seeded in a 384-well plate, 1,000 cells per well, and incubated for 2–3 h, to enable their attachment to the substrate. OT-I T-cells activated and cultured for 3 or 7 days as described above, were then added on top of the B16 cells. The entire well was imaged every 6 h, using a Hermes^® microscope (IDEA BioMedical Ltd.) equipped with automated scanning optics, high-precision autofocus, and a closed environmental chamber. The number of live (e.g., GFP expressing) B16 cells was then counted from the image, using WiSoft^® software (IDEA Bio-Medical Ltd.).