Anti cd4 apc alexafluor750

Intracellular cytokine measurement was performed according to Körholz et al., 2021 (7 (link)). Briefly PBMCs (1x10⁶/ml) were left unstimulated or stimulated with 10 ng/ml PMA (Phorbol-12-Myristat13-Acetat) and 1µg/ml Calcium-Ionophore (both Merck KGaA, Darmstadt, Germany) under addition Brefeldin A (1µl/ml, BD-Biosciences San José, CA) for 12h overnight. Cells then were harvested and washed twice with PBS/1%FCS. For surface staining, cells were incubated with anti-CD45RO-PE (5µl/test) (Biolegend, San Diego, CA), anti-CD3-APC (2µl/test), anti-CD4-APC-AlexaFluor750 (5µl/test) and anti-CD45-Krome Orange (5µl/test) (Beckman Coulter, Krefeld, Germany) for 30 min at 4°C. After beeing washed twice with PBS/1% FCS, cells were fixed and permeabilized with 100µl Cytofix/Cytoperm™ (BD Biosciences, San José, CA) for 20 min at 4°C and then washed twice according to the manufacturers instructions. For intracellular staining, cells were incubated with anti-IFNγ-FITC (5µl/test), anti-IL4-PE Cy7 (5µl/test) (both Biolegend) and anti-IL-17A eFlour 450 (5µl/test) (eBioscience/Thermo Scientific) and anti-CD4 APC-AlexaFluor750 (2µl/test) (Beckman-Coulter, Krefeld,Germany) for 45min at 4°C. Cells were washed twice with Perm/Wash Buffer (BD Biosciences) and diluted in 500µl PBS/1% FCS. Analysis was performed on a BD LSR II.

EDTA-treated whole blood was incubated using the corresponding monoclonal antibodies: anti-CCR7-FITC, anti-CD57-PE, anti-CD3-PerCP5.5, anti-CD45RA-PCy7 and anti-CD8-APC (all from BDBiosciences, New York, NY, USA); anti-CD4-APC-AlexaFluor750 and anti-HLA-DR-PB (all from Beckman Coulter, Miami, FL, USA). Proportions of T-cells were analyzed by flow cytometry using a Navios Cytometer and Kaluza Software (Beckman Coulter, Miami, FL, USA) [40 (link)].

Whole blood cytokine levels and intracellular cytokine staining were conducted on heparinized blood drawn from 10-week old South African infants. 10 milliliters of blood were taken and used both for the cytokine assays and genotyping. Samples were stimulated ex-vivo with media or BCG (strain SSI, 1.2 x 10⁶ CFU’s/ml) for 7 hours at 37°C. For whole-blood assays, IL-2 and IFN- γ were measured by multiplex bead array technology according to manufacturer’s instructions (Bio-Rad, Hercules, CA, USA) and read on a Luminex luminometer (Luminex, Austin, TX, USA). Basal cytokine levels measured in plasma harvested from unstimulated blood were subtracted from values obtained from BCG-stimulated blood [11 (link)].
For intracellular cytokine staining, Brefeldin A was added to the above samples and cells were incubated for 5 additional hours. Samples were frozen in 40% RPMI and 40% DMSO with 10% FCS and thawed prior to the time of data analysis. Flow cytometry was performed at the University of Washington Flow Cytometry Core Facility on an LSRII 5-laser flow cytometer (Becton Dickenson, Inc) using antibodies against cell surface markers including: anti-CD3 ECD (Beckman Coulter, UCHT1), anti-CD4 APC-Alexa Fluor 750 (Beckman Coulter, 13B8.2), and anti-CD8 PerCP Cy5.5 (BD, SK1). Antibodies against cytokines included anti-IL-2 PE (BD, MQ1-17H12) and anti-IFN-γ (BD, B27) [33 (link)].