Quicklysis

Immune cells carry out some of their functions by means of cell-to-cell contact and via membrane receptors or antigens that are expressed at the cellular surface. Some molecules appear at various stages of cell differentiation or activation. In the present study, we recorded the following molecules: CD4, CD8, CD19, CD56, CD71, CD97, CD134, and CD173.
Phenotypic analysis of the various cell populations was carried out by immunofluorescence by means of flow cytometry with double or triple labeling. This technique constitutes a useful tool for cell identification and characterization. Following intravenous peripheral blood collection, 50 μl of blood were incubated for 10 minutes with 3 μl of the following combinations of monoclonal antibodies (Pharmigen):

CD4-PE + CD8-FITC + CD19-Cy5

CD8-FITC + CD56-PE

CD71-FITC + CD97-PE

CD134-FITC + CDw137-PE

Afterwards, 1 mL of a hematolysis solution (Quicklysis, Cytognos) was added and the blood solution was kept in the dark for 5 minutes, after which time 10,000 cells were subjected to cytometric analysis.
Immunofluorescence analysis was carried out by means of a Vantage (Becton Dickinson) FACS flow cytometer, equipped with an argon laser (488 nm) that excites the FITC, PE, and Cy5 fluorchromes, emitting at 520, 575, and 667 nm, respectively.

To confirm the phenotype of mMSC cells were stained with specific antibodies during 30 min at 4 degrees. MSC were positive for CD29, CD44, and Sca-1 and negative for CD45, CD11b, and CD14 (data not shown).
To analyze immune infiltrated cells, tumor mass was carefully washed with HBSS 1X and mechanically processed prior enzymatic digestion with 1 mg/mL of collagenase D (Roche; Catalog #11088858001). Tumor cell suspension was counted by trypan blue and 5 x 10⁵ alive cells were labelled with specific antibodies during 30 min at 4 degrees. Red blood cells were lysed using QuickLysis (Cytognos; Catalog #CYT-QL-1) for 20 min in darkness. Used antibodies are indicated in supplementary information (Supplementary Table 1). Samples were acquired in a FacsCanto II (BD, San Jose, CA) cytometer and analyzed using FacsDiva software.

Cells were labeled with fluorescent antibodies (shown in Table 1) against human to study the phenotype of different subsets of T cells (shown in the Table 2), the early activation of T cells (CD25+ CD134+) or the exhaustion of T lymphocytes by measuring the levels of inhibitory receptors.
Cell phenotype was studied in fresh samples and after 14 days of culture using the gating strategy for identifying T cell subsets shown in Supplementary Figure S1. In the case of CAR-T cell assays, the phenotype was analyzed 7 days after transduction. Staining protocol was performed at 4°C during 30 min and erythrocytes were lysed with quicklysis (Cytognos). Events were acquired using a BD FacsCanto II cytometer, and data were analyzed with FacsDiva software (BD) and FlowJo software version 10.6.1.