Brilliant violet 421 anti mouse cd3

OX40-humanized mice were implanted with MC38 tumor cells and treated with OX40 antibodies on days 9 and 14 post implantation. On day 17, tumors were collected and dissociated into single cell suspensions by using a digestive solution (1640 medium + 2%FBS + Collagenase IV (Sigma, C5138) + DNase I (Sigma, D5025)), and spleens were ground with sterilized glass slides and filtered through a steel mesh. Red blood cells were lysed using red cell lysing buffer (TIANGEN, RT122). Single cell suspensions were first incubated with the LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (Invitrogen, L34970), then labeled with the following antibodies in flow cytometric analyses: Brilliant Violet 421™ anti-mouse CD3 (BioLegend, 100228), PE anti-mouse Ki-67 (BioLegend, 652404), PerCP anti-mouse CD8a (BioLegend, 100732), APC/Cyanine7 anti-mouse CD45 (BioLegend, 103116), PE/Cyanine7 anti-mouse IFN-γ (BioLegend, 505826), eFluor™ 450 anti-FoxP3 (Invitrogen, 48-5773-82) and eFluor™ 506 anti-CD4 (Invitrogen, 69-0042-82). Intracellular FoxP3, IFN-γ and Ki67 were labeled following the product manual. Flow cytometry analysis was performed using Cytek^® Aurora (Cytek Biosciences).

Single skin cells prepared above were stained with cell-surface antibodies for 30 min at 4 °C. Then, cell phenotyping was performed on a Cytoflex Flow Cytometer (Beckman Coulter, CA, USA). The cell-surface antibodies used were as follows: PE/Cyanine7 anti-mouse CD45 (103114, BioLegend), PerCP/Cyanine5.5 anti-mouse CD45 (103132, BioLegend), APC/Cyanine7 anti-mouse CD11b (557657, BD Pharmingen), APC anti-mouse CD64 (139306, BioLegend), PE anti-mouse Ly6C (128007, BioLegend), FITC anti-mouse Ly6G (127606, BioLegend), Brilliant Violet 421 anti-mouse CD3 (100336, BioLegend), Brilliant Violet 605 anti-mouse CD19 (115540, BioLegend), and Brilliant Violet 421 anti-mouse CD192 (CCR2) (150605, BioLegend).