Pe cyanine7 anti mouse ifn γ

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).

To determine NK cell responses in blood, spleen and lung single-cell suspensions were stained as previously described, with minor modifications.^{71 (link),72 (link)} We incubated single-cell suspensions in 1.5 mL Eppendorf tubes for 2 h at 37°C in a medium containing brefeldin A. Cells were suspended in buffer and incubated with anti-CD16/CD32 mAb for 30 min at room temperature. The cells were then stained for surface markers, fixed, permeabilized, and intracellularly stained for cytokines, according to the manufacturer’s instructions. The antibodies and reagents used for flow cytometry were as follows: PE-anti-mouse CD49b (Cat. #108907), FITC-anti-mouse CD3 (Cat. #100204), PE-anti-mouse-CD8a (Cat. #100707), APC-anti-mouse-CD4 (Cat. #100412), anti-mouse CD16/32 (Cat. #101320), APC-anti-mouse-CD107a (Cat. #121613), APC anti-mouse FceRla (Cat. #134316) and PE/Cyanine7-anti-mouse IFN-γ (Cat. #505825, BioLegend). Stained cells were analyzed by flow cytometry using a Cytoflex-LX flow cytometer (Bekeman). Data were analyzed using FlowJo software (Tree Star).