Facsaria 2 cell sorter

For measurement of mCherry fluorescence intensities, 3.0 × 10⁵ cells were resuspended in FACS buffer (PBS containing 2% FBS), filtered through a cell strainer and analyzed using a BD LSRFortessa Cell Analyzer (BD Biosciences) with BD FACS Diva software (BD Biosciences).
For the confirmation of erythroid differentiation, 1 × 10⁵ cells were stained with anti-human CD235a-FITC (#349103, Biolegend, 1:20) and CD71-APC (#334107, Biolegend, 1:20) in a total volume of 10 µL FACS buffer containing 0.5 µL Human TruStain FcX^TM (biolegend). Isotype controls were conducted using FITC and APC Mouse IgG2a antibodies (#400209 and #400221, Biolegend). After a 20 min incubation on ice, the samples were washed with 100 µL PBS containing 0.5 µg/mL DAPI. Finally, the cells were resuspended in 100 µL FACS buffer and passed through a cell strainer for analysis on a BD FACS ARIA II Cell Sorter (BD Biosciences) with the BD FACS Diva software version 8.0.1 (BD Biosciences).
For measurement of PPIX fluorescence intensities, which displays an autofluorescence with a peak at 632 nm when excited at 409 nm^{57 (link)}, the Qdot^® 605 filter setting (excitation 405 nm, emission 610 nm + /− 20 nm) of the BD FACS ARIA II Cell Sorter was used.
Flow cytometry data were analyzed by FlowJo software v9.7.6 or higher (Tree Star).

C-Kit-D816V-stimulated T cells were plated by limiting dilution (0.5–2.0 cells/well) and expanded in a 96-well U-bottom plate (Becton, Dickinson and Company) in AIM-V medium supplemented with 5% heat-inactivated human serum, 40 ng/mL anti-CD3 antibody (Clone UCHT 1; Becton, Dickinson and Company), and 120 IU/mL IL-2 (PeproTech, Inc.). The expanded clones were examined for IFNγ production after peptide stimulation, and a C-Kit-D816V-specific T cell clone was obtained. A single T cell from the obtained clone was sorted into a 96-well plate (4titude, Wotton, UK) by the FACSAria II cell sorter (Becton, Dickinson and Company). PIK3CA-H1047R specific T cells were enriched by IFNγ secretion assay (Miltenyi Biotec, Auburn, CA, USA), followed by sorting of a single T cell into a 96-well plate (4titude) by the FACSAria II cell sorter (Becton, Dickinson and Company).
The sequences of TCR genes of the sorted T cell clones were determined, as reported previously [33 (link)]. The cDNA of TCRα and TCRβ chain was amplified from single cells and sequenced at Eurofin Genomics K.K. (Tokyo, Japan).

Mouse tumor single-cell suspensions were generated as described in the previous section. Cells were stained with anti-CD45-Alexa-Fluor-780 and anti-CD3-APC for flow cytometry sorting for CD3⁺ T cells (CD45⁺CD3⁺) on FACSAria II Cell Sorter (BD). Dead cells were excluded using DAPI. The purity of flow-sorted populations was above 95%. 2.5 × 10⁵ sorted CD3⁺ T cells from the spleen of C57BL/6 mouse were stimulated on plates coated with 2 μg/ml anti-CD3 antibody (Biolegend) and soluble 1 μg/ml anti-CD28 antibody (Biolegend) in the T-cell medium for 8 h. Following indicated incubation, cells were harvested and suspended in TriZol Reagent (Invitrogen) for subsequent RNA isolation, and supernatants were stored at −80°C immediately and used for cytokine assay. For sorting CD8⁺ naïve T cells, spleen cells of B6 mouse were stained with anti-CD45A-Alexa-Fluor-780, anti-CD3-APC, anti-CD8-PE antibodies for flow cytometry and CD45A⁺CD3⁺CD8⁺ cell were sorted on FACSAria II Cell Sorter (BD). Dead cells were excluded using DAPI. The purity of flow-sorted populations was above 95%.

For isolating γδT cells in breast tissue, fresh specimens were thoroughly washed twice in PBS containing 10× penicillin and streptomycin (Invitrogen), cut into small pieces (1 mm³) with sterile scalpel blades and digested in RPMI 1640 (Invitrogen) medium supplemented with 2% FBS, 1 mg/mL type IV collagenase and 1 mg/mL hyaluronidase (Sigma) for 4 h at 37 °C to obtain a single-cell suspension. Tissue debris was removed by passing the suspension through a 100-μm filter. CD4+ Tregs (CD3+CD4+CD25+), Vδ1 T cells (CD3+Vδ1+), Vδ2 T cells (CD3+Vδ2+) and CD73+Vδ1 T cells (CD3+CD73+Vδ1+) were sorted by a FACS Aria II cell sorter (BD Biosciences). For isolating CD3+, Vδ1 T and Vδ2 T cells in the peripheral blood from healthy donors, peripheral blood mononuclear cells (PBMCs) were obtained with Ficoll (1.077 g/ml) density-gradient centrifugation at 400 × g for 15 min. Then, the cell pellet was resuspended in PBS, labelled with corresponding antibodies and processed with a FACS Aria II cell sorter (BD Biosciences) to separate the different populations. The purity of all the sorted cells was >90%.

Clonal subpopulations are generated from parental cell lines by sorting one single cell per well into 96-well plates using a FACSAria II cell sorter (BD Bioscience). Single cell-derived populations are subsequently allowed to proliferate for expansion. A single expanded clone is used for both control and co-transfection with the Cas9/GFP and sgRNA vectors. Select cell populations were seeded into 12-well plates overnight before transfection with 500ng pCas9_GFP and 500ng sgRNA expressing plasmids using FugeneHD (Roche). 48 hours after transfection, successfully transfected single cells are isolated by FACS sorting for GFP-positivity using a FACSAria II cell sorter (BD Bioscience) followed by recovery and expansion in 12-well plates for 2–3 days. At confluency, cells were collected for a second round of FACS sorting and single GFP-negative cells were sorted into individual wells in a 96-well plate to ensure that random Cas9/GFP integration did not occur. Following clonal expansion editing is validated using Sanger sequencing and phenotype verification is performed.
To generate luciferase/GFP-positive populations, cells were infected with lentivirus generated from pLV-Luc-IRES-GFP viral plasmids (a generous gift from Dr. Robert Weinberg’s lab) and then sorted for GFP-positive populations.