Anti mcd4 brilliant violet 510 clone gk1.5

Control WT (CD4-Cre alone) and Pik3ip1^fl/fl × CD4-Cre mice were infected intravenously with 15,000 CFU L. monocytogenes in 200 µl PBS. Bacterial titers were quantified by lysing whole livers in PBS, plating a 1:10 dilution on brain-heart infusion agar plates, and culturing overnight. Cells were stained with the following antibodies and dyes and analyzed by flow cytometry: Ghost Red 780 viability dye (Tonbo Biosciences; 13-0865-T100); anti-mCD4 Brilliant Violet 510 (clone GK1.5; BioLegend, 100449); anti-mCD8a PE (clone 53-6.7; Tonbo Biosciences, 50-0081-U100); anti-mCD62L PerCP-Cy5.5 (clone MEL-14; eBioscience, 45-0621-80); anti-mKLRG1 FITC (clone 2F1; Tonbo Biosciences, 35-5893-U100); anti-mCD127 PE-Cy7 (clone A7R34; Tonbo Biosciences, 60-1271-U025); and anti-mCD44 violet Fluor 450 (clone IM7; Tonbo Biosciences, 75-0441-U025). All Ab staining was performed at 4°C in PBS containing 1% BGS and 0.1% sodium azide. GP33 tetramers were provided as APC fluorophore-conjugated tetramers by the NIH tetramer core facility and used for identification of gp33-specific CD8⁺ and CD4⁺ T cells.

Splenocytes and lymphocytes obtained from WT and conditional TrIP KO mice were stimulated in complete RPMI with 3 µg/ml biotinylated anti-CD3 and anti-CD28 in the presence of 15 µg/ml streptavidin (anti-mCD3ε biotin, clone 145-2C11; Tonbo Biosciences, 30-0031-U500; anti-mCD28 biotin, clone 37.51; BD Biosciences, 553296; streptavidin; Millipore Sigma, 189730). Cells were activated for 15, 30, or 60 min or 4 h. Activation was stopped with 1 ml cold PBS, followed by centrifugation, aspiration of media, and lysis or fixation.
The following antibodies and dyes were used for flow cytometry: anti-mCD4 Brilliant Violet 510 (clone GK1.5; BioLegend, 100449); anti-mCD8α v450 (clone 53-6.7; Tonbo Biosciences, 75-0081-U100); anti-pS6 (S235/236) Alexa Fluor 647 (clone D57.2.2E; Cell Signaling, 5316S); anti-mCD69 FITC (clone H1.2F3; BD Biosciences, 557392); and Ghost Dye Red 780 (Tonbo Biosciences; 13-0865-T100).