Live dead fixable dead cell stain kit lda

IFN-γ and TNF-α secreting, and FoxP3+, CD4+ T-cells in infant PBMC were identified by ICS (intracellular cytokine staining). PBMC were washed with media, and then left unstimulated or stimulated for 4 h with PMA/Ionomycin (BD Biosciences). The stimulations were done in the presence of 1 μl Brefeldin A (BD Biosciences). Cells were first stained with surface Ab to CD45RO (clone UCHL1), fixed and permeabilized with the FoxP3 buffer set (BD Biosciences), and then stained with Abs to CD3 (clone UCHT1), CD4 (clone SK3), CD8 (clone SK1), IFN-γ (clone B27), TNF-α (clone 6401.1111), and FoxP3 (clone 259D/C7) (all Abs from BD Biosciences). Cells were analyzed using a FACSAria flow cytometer (BD Biosciences). LIVE/DEAD^® Fixable Dead Cell Stain Kit (LDA) (Invitrogen) was used to exclude nonviable cells from analysis. Relevant cells were identified as LDA−/CD3+/CD4+/CD8−/CD45RO+ or CD45RO−/IFN-γ+ or TNF-α+ or FoxP3+ cells (Supplementary Fig. 1). Data was analyzed using FlowJo^® software (Treestar).

Infant PBMC from the first study visit were used for intracellular cytokine staining. PBMC were washed with media, and then left unstimulated or stimulated with 1 µM R-848 (Invivogen) ×16 h. The stimulations were done in the presence of 1 µl Brefeldin A (BD Biosciences) ×16 h. Cells were stained with LIVE/DEAD® Fixable Dead Cell Stain Kit (LDA) (Invitrogen), fixed and permeabilized with Cytofix/Cytoperm™ (BD Biosciences), and stained with Abs. Monocytes were identified as LDA-/CD1c-/CD19-/CD36+/CD123-/CD303- and myeloid DCs were identified as LDA-/CD1c^hi/CD19-/CD36-/CD123-/CD303- (all Abs from eBiosciences). TNF-α, IL-6, and pro-IL-1β production was measured by staining with the respective mAbs (BD Biosciences). In some infant PBMC, surface expression of the leptin receptor (ObR) was determined by staining with an anti-ObR mAb (BD Biosciences). Cells were analyzed using a FACSAria™ flow cytometer (BD Biosciences). Data was analyzed using FlowJo® software (Treestar).