Live dead violet dye

Peripheral blood mononuclear cells were collected by leukapheresis as previously described and purified using Lymphoprep (Stemcell Technologies, Vancouver, BC) gradient, then frozen in liquid nitrogen prior to thawing and analysis (13 (link)). Phenotypic markers of MDSCs were evaluated by flow cytometry with a lineage-marker negative population gated to exclude CD3-, CD19-, or CD56-expressing cells using antibodies to CD11b, CD14, HLA-DR, and CD33 from BD Biosciences (San Jose, CA), except where indicated. The lineage-marker positive cells (CD3⁺, CD19⁺, and CD56⁺) highly expressed HLA-DR, which was used as a reference to set the HLA-DR^low gate which included the cells below the bottom edge of the clearly positive expression of that molecule from the lineage positive cells. Peripheral blood mononuclear cells were stained with Live/Dead violet dye (Invitrogen, Carlsbad, CA) to gate on live cells. Data were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed with Flowjo software (TreeStar, Ashland, OR). Data were analyzed by the principal investigator, J.S.W., and biostatisticians X.Z. and Y.A.C.

After thawing PBMCs and washing twice with RPMI medium contained 10% FBS (R10) (Gemini), cell number and viability were calculated. A total of 2×10⁶ PBMCs were plated in 96-well round-bottom plates in R10. After centrifugation and removal of media, cells were surface stained with LIVE/DEAD violet dye (Life Technologies, Grand Island, NY) for 15 minutes at room temperature. Cells were washed with PBS, then stained with a surface stain cocktail mix for 30 minutes at 4⁰C. Following cell surface staining, cells were permeabilized with the FOXP3/Transcription Factor and Fixation/Permeabilization buffer according to the manufacturer’s recommendations (eBioscience, San Diego, CA) for intranuclear staining or Cytofix/Cytoperm buffer (BD Biosciences, San Jose, CA) for intracellular staining. Afterwards, intranuclear or intracellular cytokine staining was performed. Lastly, cells were fixed with 1% paraformaldehyde (BD Biosciences) and acquired on a LSRII flow cytometer (BD Biosciences).

Cultured cells were stained with 50uL of LIVE/DEAD violet dye (Life Technologies, Grand Island, NY) in PBS for 15 minutes at room temperature, to exclude dead cells from the analysis. Next, cell surface antigens were stained with a cocktail mix consisting of titrated volumes of anti-CD3 APC-Cy7, anti-CD4 BV785, anti-CD8 AlexaFluor 700, and anti-CXCR5 PE-Cy7 for 30 minutes at 4°C. Following cell surface staining, cells were treated with cytofix/cytoperm (BD Biosciences) according to the manufacturer’s recommendations. Intracellular staining was then performed for 30 min at 4°C using the following conjugates: anti- IFN-γ FITC, anti-IL-17A PerCP-Cy5.5, anti-IL-2 APC, and anti-IL-4 PE-Dazzle 594. Anti-IL-2 APC antibody was purchased from BD Biosciences, while other cytokine fluorescent antibodies were purchased from Biolegend. Cells were fixed with 1% paraformaldehyde (PFA) and acquired on a BD LSRII flow cytometer (BD Biosciences).

Tumors were harvested when they reached approximately 10–15mm in the largest diameter and were mechanically disrupted and subjected to enzymatic digest using a 1mg/mL collagenase D solution (Roche). Red blood cells were lysed using a 0.84% NH₄Cl solution. Cells were washed twice with RPMI+10%FBS and passed through a 70-μm cell strainer. The resultant cell suspension was washed twice with ice cold PBS and stained with LIVE/DEAD-Violet dye according to manufacturer’s recommendation (Life Technologies). Fc-receptors were blocked using 1μg anti-CD16/CD32 (Biolegend; TruStain FcX). Staining for surface antigens was performed in PBS+1%BSA using the following antibodies: CD45 (Biolegend; clone 30-F11), F4/80 (Biolegend; clone BM8), MHCII (Biolegend; clone M5/114.15.2), CD80 (BD Biosciences; San Jose, CA; clone 16-10A1), CD86 (BD Biosciences; clone GL1), Gr-1 (Biolegend; clone RB6-8C5), CD11b (Biolegend; clone M1/70), NK1.1 (eBioscience; San Diego, CA; clone PK136), CD3 (Biolegend; clone 145-2C11), CD4 (eBioscience; clone RM4-5), CD8a (Biolegend; clone 53-6.7), CD69 (Biolegend; clone H1.2F3), CD206 (Biolegend; clone C068C2), FoxP3 (eBioscience; clone NRRF-30), and HER2 (BD Bioscience; clone Neu 24.7). Intracellular staining for Foxp3 was performed using fixation and permeabilization buffers according to the manufacturer’s recommended protocol (eBioscience).