Cd19 pe cf594

After 6 days culture, cultured cells were analyzed for expression of surface markers by multi-color flow cytometry. Cell aliquots were treated with anti-human Fc mAb for 20 minutes and stained for 30 minutes with selected combinations of fluorochrome-conjugated antibodies.
For the analysis of B cell subpopulations and activation markers, the monoclonal antibodies used were CD19-PE-CF594 (BD Biosciences, San Jose, CA), CD62L-PE-Cy5 (BD Biosciences), IgD-FITC (BD Biosciences), CD27-PE-Cy7 (BD Biosciences), CD24-PE (BD Biosciences), CD38-PerCP-Cy5.5 (Beckman Coulter, Brea CA), CD138-PE-Cy7 (eBioscience, San Diego, CA), CD23-PE (BD Biosciences), CD21-PE-Cy5 (BD Biosciences), IgG-PC7 (BD Biosciences), CD86-Alexa 700 (BD Biosciences) and CD95-Pacific Blue (BioLegend, San Diego, CA).
For plasma cell staining, cultured cells were first incubated with monoclonal antibodies to surface markers (CD38, CD138 and CD19), fixed and permeabilized with Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) and then incubated with Blimp-1-PE (R&D systems) and Pax5-APC (eBioscience) for 30 minutes.
For the proliferation assay, 1–10×10⁶ cells of interest were labeled with 1μM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) before culture initiation. After 6 days of culture, cells were harvested, incubated with antibodies, and dilution of CFSE was assessed by flow cytometry.

Tumor cells isolated from mice were thawed and stained with LIVE/DEAD Fixable Violet Dead Staining Kit (ThermoFisher). Subsequently, the cells were divided and stained with cocktails of fluorochrome-conjugated monoclonal antibodies: CD3 PE-CF594, CD19 PE-CF594, CD49b PE-CF594 (all from BD Biosciences), CD45 BV605, CD11b PerCP-Cy5.5, CD11c BV650, F4/80 AlexaFluor 700, Ly6C PE, Ly6G APC-Cy7, MHC II FITC, CD80 PE-Cy7 (all from Biolegend) for myeloid cell identification and CD45 BV605, CD3 BV650, CD4 FITC, CD8 APC-Fire, CD25 PE, CD44 PE-Cy7, CD62L PerCP-Cy5.5 (all from BioLegend) for lymphocytes identification. Then, the cells were fixed using FoxP3 Fixation Permeabilization Staining Kit (eBioscience). Tumor cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or FoxP3 APC (eBioscience) antibodies, respectively. The analysis was performed using FACSFortessa flow cytometer with Diva software (Becton Dickinson).

Eight to ten-week old female C57BL/6 mice were subcutaneously inoculated in the right flank with MC38/0 cells (1.1 × 10⁶/0.2 ml/mouse). On the 14th, 15th and 17th day of the experiment, mice were injected i.t. with LVs encoding shRNA against IL-10 (shIL10–3, 2x10⁶TU/50 μl/mouse) or reference LVs encoding scrambled shRNA against human GAPDH (shN). Two days after the third injection, the mice were sacrificed and their tumor nodules were dissected and homogenized. Efficacy of transduction in tumors was measured by flow cytometry as the fluorescence intensity of EGFP among cells isolated from tumors. Concentration of IL-10 was estimated by ELISA in supernatants collected from 24 h culture of 5 mg tumor tissue/ml. Myeloid and lymphocyte populations in tumors were analyzed using LSR Fortessa with Diva software (Becton Dickinson) after staining with fluorochrome-conjugated antibodies: CD45 V500, CD3 PE-CF594, CD19 PE-CF594, CD49b PE-CF594 (all from BD Biosciences), CD11b PerCP-Cy5.5, CD11c BV650, F4/80 AlexaFluor 700, Ly6C BV510, Ly6G BV605, MHC II APC-Cy7, for myeloid cell identification (all from Biolegend) and CD45 BV605, CD3 BV650, CD4 FITC, CD8 APC-Fire, (all from BioLegend) for lymphocyte identification.