Fixable blue dead cell stain kit

Single-cell suspensions from the spleen or liver (after lymphocyte isolation on a 37.5%-67.5% Percoll gradient) or small intestine (after digestion with collagenase VIII from Sigma and lymphocyte isolation on a 40–100% Percoll gradient) were incubated with Fc blocking antibody (2.4G2) and the fixable blue dead cell stain kit (Invitrogen). Surface molecules were stained using antibodies against CD45.2 (104), CD3 (145-2C11), CD19 (1D3), NK1.1 (PK136), CD49a (Ha31/8), CD11b (M1/70), CD27 (LG.3A10), CD43 (S7), KLRG1 (2F1), Ly49G2 (4D11), CD69 (H1.2F3), CD11b (M1/70), MHCII (M5/114.15.2), Ly6C (AL-21), CD3 (145-2C11), CD19 (1D3) from BD Biosciences; NKp46 (29A1.4), CD49b (DX5), NKG2A (16a11), Ly49D (4E5), Ly49H (3D10), and F4/80 (BM8) from eBioscience and CD11c (N418) and Ly6G (1A8) from BioLegend. For intracellular staining, cells were fixed and permeabilized with an intracellular staining kit (eBioscience) and the following antibodies were used: anti–GR XP rabbit mAb (D8H2) and rabbit mAb IgG XP (DA1E) from Cell Signaling Technology; anti–IFN-γ (XMG1.2) from BioLegend; anti–Rorγt (Q31-378), anti-TNF (MP6-XT22), anti–granzyme B (GB11), anti-Ki67 (B56), and anti–IL-10 (JE5-16E3) from BD Biosciences; and anti-Eomes (Dan11mag) from eBioscience.

C57BL/6J mice were treated with either 0.35 mg of anti-CD4 monoclonal antibody (clone YTS 191, BioXcell), 0.1 mg anti-mouse CD8β monoclonal antibody (Lyt 3.2, BioXcell) or 0.85 mg of isotype control rat IgG2b (clone TNP6A7, BioXcell). Doses were given on day −3, −1 prior to challenge and day +1 post sporozoite challenge, 100 μL i.p. To assess the efficacy of cell depletion, single-cell suspensions from perfused livers were obtained as previously described (Mastelic et al., 2012 (link); Tupin and Kronenberg, 2006 (link)). Cells were stained with the following antibodies: Brilliant Violet 421™ anti-mouse CD3 (1 in 200 dilution, Biolegend®), PE anti-mouse CD8 (1 in 400 dilution, Biolegend®), APC anti-mouse CD4 (1 in 400 dilution, Biolegend®) and the Fixable Blue Dead cell stain kit (1 in 200 dilution, Invitrogen™, L34961). Cells were acquired using a BD LSRFortessa™ flow cytometer and BD FACSDIVA™ software. Data were analysed using FlowJo™ software (Tree Star).

Approximately five million freshly isolated PBMCs per sample were used for staining. For the first eight patients, we used PE-Labeled Human CD19 (20–291) Protein (Acro Biosystems). Later, anti-FMC63-FITC antibody by Acro Biosystems was used. Comparison of staining with protein CD19 and anti-FMC63 antibody is shown in Supplementary Figure S1. Besides detection of CAR-T cells the samples were stained with antibodies against antigens CD3, CD4, CD8, CD45RA, CD62L, CD27, CD28, CD57, PD-1, TIM-3, and TIGIT. A second detection panel against antigens CD3, CD4, CD8, CD14, CD16, CD19, CD45, CD56, and TCRgd was used to determine significant leukocyte subsets in the samples. Antibody panels used in this study were used previously (20 (link)) and can be found in Supplementary Tables S1, S2. All antibodies were titrated before use, and fluorescence-minus-one controls for selected antibodies were measured. Firstly, PBMCs were stained using a fixable blue dead cell stain kit (Thermo Fisher Scientific, United States), washed with FACS buffer, and then stained with antibody mix in Brilliant Stain Buffer (BD) at room temperature for 30 min. The samples were washed with FACS buffer prior to measurement.

Using a single panel of 23 antibody-fluorochrome conjugates and one live/dead stain (fixable blue dead cell stain kit (Thermo Fisher, L34961) T cell, B cell, NK cell and monocyte subsets were identified (Figure 1; Supplementary Table 1). Single cells were gated using forward scatter area and forward scatter height. Lymphocytes and monocytes were gated using side scatter and forward scatter followed by exclusion of dead cells using the live/dead stain (Figure 1). Using CD3 and CD19 three main subsets were classified including CD3+ (T cells) CD19+ (B cells) and CD19-CD3- (NK cells and monocytes).

Mast cells were isolated from 6 mm biopsies obtained from 6 mm skin punch biopsies acquired from the buttocks of volunteers with and without psoriasis under local anaesthesia in the Salford Royal Foundation Trust Hospital Dermatopharmacology Unit. Biopsies were stored in ice-cold PBS. Skin biopsies were cut into small pieces in digestion solution containing RPMI 1640 medium (Sigma-Aldrich, Gillingham, UK), 0.5% BSA (ThermoFisher Scientific), 100 U.mL⁻¹ penicillin and 100 µg.mL⁻¹ streptomycin, 0.5 Wunch units.mL⁻¹ of Liberase TM (Roche, Basel, Switzerland), and 100 ng.mL⁻¹ of SCF (Genscript, Piscataway, NJ, USA), then incubated overnight at 37 °C, 5% CO₂.
Digested skin was passed through a 40 μm mesh cell strainer and cell concentration was adjusted to 50 × 10⁶ cells.mL⁻¹. Cell suspension was stained for 15 min in PBS 0.5% BSA, 2 mM EDTA with CD45 (2D1), Human Lineage cocktail (CD3 (UCHT1), CD14 (HCD14), CD19 (HIB19), CD20 (2H7) and CD56 (HCD56)), CD117 (104D2), FcɛRIα (AER-37) antibodies (all from BioLegend, San Diego, CA, USA), and a viability dye (Fixable Blue Dead Cell Stain Kit, ThermoFisher Scientific). CD45+, Lin-, CD117+, FcɛRIα+, and MCs were sorted using a BD Influx (BD Biosciences) (Supplementary Figure S3), collected in 1000 cell aliquots and stored at −80 °C until required.