Cd3 bv570

The procedure of flow cytometric analysis of CMV- and IAV-specific CD8⁺ T cells prior to and post in vitro stimulation was described [40 (link)]. Briefly, freshly PMBCs were stained with fluorescently labeled dextramers or tetramers specific to CMV-pp65 (NLVPMVATV) and IAV-M1 (GILGFVFTL) (Immudex, Copenhagen, Denmark and NIAID tetramer core) first at room temperature for 20 min; followed by staining of cell surface markers including antibody against CD3, CD8, CD62L, CD45RA, CD95, CD27, CD28 CD70, CD127, and CD69 at 4°C for 30 min. Cells were washed again with FACS buffer (Hanks solution with 0.3% Sodium Azide), and then fixed immediately with 3% formaldehyde and 1% FBS in FACS buffer. Fixed cells were further stained with intracellular markers (perforin and granzyme B) at 4°C for 30 min. Stained cells were collected by BD_Symphony and were analyzed using FlowJo version 7.6.5 software.
Antibodies (CD3-BV570, CD62L-FITC, CD62L-PE-Cy7, CD45RA-PE-Cy7, CD45RA-APC, CD95-PE-Cy5, CD27-PE, CD28-BV785, CD28-PerCP-Cy5.5, CD70-PE, CD127--BV711, CD69-BV650, and granzyme B) were purchased from Biolegend, and antibodies against CD8-BUV496, CD27-BUV395, and perforin-PE-CF594 were purchased from BD.

The presence of immune subsets in peripheral blood, spleens and brains was assessed in adult and 3- to 4-day-old pups by flow cytometry. All animals were anaesthetized and perfused with saline in order to flush out circulating blood cells from the organs. Spleen cells were collected by homogenization and filtering through a 40-μm cell strainer into FACS buffer (PBS with 0.5% BSA). Brains were harvested in FACS buffer followed by three sequential digestion steps in RPMI+25 mM HEPES+Collagenase D (1 mg ml⁻¹)+DNAase 1 (0.2 mg ml⁻¹) at 37 °C for 5 min each. Spleen and brain single-cell suspensions were counted using a Z2 Coulter Counter (Beckman Coulter) and subsequently stained with a 10-colour panel with the following antibodies: Ly6G-FITC, Gr1-PE, NK1.1-PerCP-Cy5.5, CD11c-PE-Cy7, CD11b-APC (all from BD Biosciences) and CD45-APC-Cy7, CD3-BV570, B220-BV650, CD4-BV711 and CD8-BV780 (all from BioLegend). Samples were acquired within an hour after staining in an LSR-II flow cytometer (BD Biosciences) and analysed on FlowJo 9.7.6 software.

Immune-phenotypic characterization of CAR-T products was performed by Flow Cytometry, using the BD FACSymphony^TM Flow Cytometer. Antibodies used in the panels tested included: Viability stain dye (Invitrogen), CD3 BV570 (Biolegend), CD4 BUV563 (BD), CD8 BUV496 (BD), CD19CAR FITC (Acro Biosystem), CD45RO BUV395 (BD), CD27 BV421 (BD), CCR7 BUV805 (BD), 41BB BV650 (BD), TCF7 PE (BD) and Ki67 PE-Cy5 (ThermoFisher). Flow Cytometry data were analyzed by FlowJo v. 10.7. The gating strategy consisted of a set of steps starting from lymphocytes, based on SSC-A and FSC-A; single cells, based on FCS-H and FSC-A; and live cells, based on MFI for Viability dye. Consecutively specific gating was done in each population of interest e.g. CD4+ T cells, CD8+ T cells, etc. Lastly, populations of interest were concatenated and exported for unbiased clustering analysis, which was done using the Rphenograph package (https://github.com/jacoblevine/PhenoGraph). Projection of the density of cells expressing markers evaluated (above mentioned) was visualized using a non-linear dimensionality reduction technique, Uniform Manifold Approximation, and Projection (UMAP). All correlations were done using Spearman correlation. All statistical comparisons were done using the Mann–Whitney U test. Statistical analysis was performed using Graph Pad Prism version 8.

Therapeutic anti-CTLA-4 (clone 9H10) antibody and isotype control antibody was purchased from BioXcell (cat: BE0131 and BE0087). Antibodies used for flow cytometry were purchased from the following sources (dilutions are indicated in parentheses): eBioscience (CD45.2 Alexa Fluor 700, cat: 56-0454 (1:200), CD3 PE-Cy7, cat: 25-0031 (1:200), CD4 ef450, cat: 48-0041 (1:200), CD4 APC-efluor780, cat: 47-0041 (1:400), CD8 PerCP-efluor710, cat: 46-0083 (1:200), CD11b APC-efluor 780, cat: 47-0112 (1:600), ICOS PE, cat: 12-5985 (1:200), ICOSL PE, cat: 12-5985 (1:200), CTLA-4 PE, cat: 12-1522 (1:200), NK1.1 PE, cat: 12-5941 (1:200), IFNγ PE, cat: 12-7311 (1:200), FoxP3 Alexa Fluor 700, cat: 56-5773 (1:100), FoxP3 APC, cat: 17-5773 (1:200), GATA-3 PE, cat: 12-9966 (1:100), RORγT PerCP-efluor710, cat: 46-6981 (1:100), Tbet PE-Cy7, cat: 25-5825 (1:100), EOMES efluor 450, cat: 48-4875 (1:100), PD-1 PE-Cy7, cat: 25-9985, (1:200)), Biolegend (CD3 BV570, cat: 100225 (1:100), CD11b BV570, cat: 101233 (1:50), Bcl-6 Alexa 594, cat: 648308 (1:50)), Invitrogen (Granzyme B PE-Texas Red, cat: GRB17 (1:125), Granzyme B APC, cat: GRB05 (1:125)) and BD Pharmingen (Ki-67-Alexa Fluor 488, cat: 561165 (1:50), CXCR5-biotin, cat: 551960 (1:100)).