Cd86 alexa 700

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.

Primary antibodies against LAMP1, LAMP2, Cathepsin D, Cathepsin L, p62, galectin-3 and secondary goat anti-mouse IgG (Alexa Fluor 647) were purchased from Abcam (Cambridge, UK). LC3A/B was from Cell Signaling (Bioke, Leiden, The Netherlands), actin-HRP from Santa Cruz Biotechnology (Santa Cruz, CA, USA), CD86-Alexa700 and HLA-DR-PeCy5 from BD Biosciences (Erembodegem, Belgium) and CD80-BV650, CD14-FITC and CD163-Alexa647 were bought from Biolegend (ITK diagnostics, Uithoorn, The Netherlands). NDP52 (CALCOCO2), secondary goat anti-rabbit IgG (Alexa Fluor 647) and HRP-conjugated antibodies reactive with mouse and rabbit were purchased from Thermo Fisher Scientific (Merelbeke, Belgium).

PBLs and cancer cells from direct and indirect co‐cultures were used for multi‐marker flow cytometry to detect the expression of immune checkpoint markers. Cells were washed and resuspended in 100 μl of staining buffer containing Human Fc Block™ (564219, BD Biosciences). The expression of immune checkpoint receptors was determined using the following antibodies: PD‐1 PE‐Dazzle 594 (329940, Biolegend), VISTA BV421 (566750, BD Biosciences), CTLA‐4 BV786 (563931, BD Biosciences), TIM‐3 BV650 (565565, BD Biosciences), LAG‐3 PE (565617, BD Biosciences), TIGIT BUV395 (747845, BD Biosciences) and CD8 BV510 (563919, BD Biosciences). In parallel, we determined the expression of respective ligands: CD80 BV510 (740150, BD Biosciences), CD86 Alexa700 (564544, BD Biosciences), PD‐L1 PE‐Cy7 (558017, BD Biosciences), PD‐L2 BV786 (563843, BD Biosciences), VISTA BV421 (566750, BD Biosciences), MHC‐II BV650 (564231, BD Biosciences), GAL‐9 PE (565890, BD Biosciences) and PVR BUV395 (748272, BD Biosciences). Flow cytometry was performed by recording 50 000 events/sample using the LSRFortessa X‐20 instrument and FlowJo V10.7.1 software (BD Biosciences).