Fitc anti human cd4 antibody

Cryopreserved CD3+ Pan T cells (PB009-1F-C-5M), CD19+ B cells (PB010-P-F-C), CD14+ monocytes (PB-011-P-F-1-C), and CD56+ natural killer cells (PB012-P-F-C) were purchased from AllCells (Shanghai, China). The CD3+ Pan T cells were stained with APC/Cyanine7 anti-human CD3 Antibody (300425, BioLegend), FITC anti-human CD4 Antibody (300506, BioLegend) and PE anti-human CD8 Antibody (344705, BioLegend) following the manufacturer’s instructions. Then, the Pan T cells were sorted into CD4+ T cells and CD8+ T cells with a BD FACSAria III instrument. CD14+ monocytes were stained with PE anti-human CD14 Antibody (301805, BioLegend) and APC anti-human CD16 Antibody (302011, BioLegend) following the manufacturer’s instructions. CD14+ monocytes were further separated into CD16+ monocytes and CD14+ monocytes (CD16-) with a BD FACSAria III instrument.
For fresh PBMCs, venous blood was collected from a healthy donor into a plastic blood tube spray-coated with K₂EDTA (367863, Becton Dickinson, NJ, USA). Blood was dissolved in an equal volume of 1 x DPBS solution and added to a SepMate™-15 tube (86415, StemCell Technologies, Canada) containing Histopaque-1077 (10771-100 ml, Sigma-Aldrich, MO, USA). After centrifugation (1200 x g, 10 min), PBMCs were collected, washed twice, and resuspended with a 1 x DPBS solution.

Activated Caspase-1 was detected with the Pyroptosis/Caspase-1 Assay Kit (#9158) from ImmunoChemistry Technologies (CA, USA) according to the manufacturer’s instructions. CD4⁺ T cells were incubated with FLICA solution at 37 °C for 1 h. CD4⁺ T cells were then incubated with FITC anti-human CD4 antibody (357,406) from BioLegend (CA, USA) for 30 min. Fluorescently labeled cells were immediately detected by flow cytometry (Agilent, Novocyte 2060R).