Anti cd19 pe antibody

Fractionation of collected blood samples for neutrophils, monocytes, DC, NK cells, T cells and B cells was performed as previously described [29 (link)]. Briefly, whole blood samples (90 mL) underwent Ficoll separation to yield PBMC and polymorphonuclear cell (PMN) populations. Single cell suspensions of PBMC or PMN were subjected to magnetic bead separation. T cells, monocytes, and neutrophils were enriched by positive selection using directly conjugated anti-CD3, anti-CD14, and anti-CD15 microbeads (Miltenyi Biotec), respectively. B cells were enriched by positive selection using anti-PE beads after staining with anti-CD19-PE antibody (Miltenyi Biotech). NK/mDC were enriched by negative selection using Streptavadin microbeads (Miltenyi Biotec) after staining with biotinylated anti-CD19, anti-CD15, anti-CD14, and anti-CD3 antibodies (eBioscience). MACS enriched cells were stained with 7-aminoactinomycin D (7-AAD), CD11c-FITC, CD15-APC and CD56-PE-Cy7 (BD Biosciences), as well as CD19-PE, CD3-VioBlue, and CD14-VioGreen (Miltenyi Biotec), and were subjected to fluorescence-activated cell sorting (FACS) on a BD FACSAriaIII flow cytometer (BD Biosciences). Cell purity of ≥98% was confirmed after the sorting procedures. Purified immune cells (≥0.5x10⁶) were frozen in homogenization buffer (Maxwell 16 LEV simply RNA kit, Promega) and stored at -80°C.

For immunofluorescence staining, cells were fixed in 4% PFA-PBS for 10 min. After washing 3× with PBS, cells were blocked with 3% BSA diluted in PBS for 1 h at RT and then incubated with anti-CD-19-PE antibody (1:50) in 3% BSA-PBS (Miltenyi Biotec) for 1 h at RT. The nuclear stain Hoechst 33258 (2 μg/ml; Sigma-Aldrich) in PBS was added for 10 min. Fluorescent images were obtained using a fluorescence microscope Leica DMi8 (Leica). To determine the number of CD19⁺ cells relative to the total number of cells, the total integrated density of the CD19 antibody was calculated and divided by the integrated intensity of the nuclei staining using ImageJ.