Lsr fortessa cell sorter

Identification of cardiomyocyte markers via flow cytometry was performed following trypsin detachment of cells from culture plates, after which cells were fixed in 4% paraformaldehyde (PFA) and permeabilized in 0.02% Tween-20, 0.5% DMSO in PBS. Blocking was then performed with 5% bovine serum album in permeabilization buffer. The cells were stained with cardiac troponin T antibody (Abcam, Cat#Ab8295) and goat anti-mouse Alexa 647 secondary antibody (Abcam, Cat#Ab150115). The cells were analyzed in a BD LSR Fortessa cell sorter using FlowJo software.

Fifteen CF and five non-CF human whole blood samples were obtained from convenience samples with available extra blood, stained, and fixed the same day blood was drawn. A master mix of brilliant stain buffer, antibodies (CD1c, CD3, CD4, CD8, CD11c, CD14, CD15, CD15, CD19, CD33, CD38, CD45, CD56, CD123, CD141, HLA-DR [Biolegend, BD Horizon, BD Pharmingen]) and whole blood was incubated for 30 minutes at room temperature. RBC lysis solution was then added for 10 minutes. The cells were washed with stain buffer and centrifuged for 5 minutes at 400g. After the final spin, the cells were resuspended in a small amount of stain buffer. For this multicolored panel, compensation beads were used for positive and negative staining controls. Data were acquired via a BD LSR Fortessa cell sorter with five lasers (blue, violet, ultraviolet, red, and green). Approximately 500 thousand events were recorded per sample. Samples were analyzed using FlowJo (v10.7.1). Each sample was analyzed and gated following the same gating hierarchy. After file concatenation, data dimensionality reduction was performed using t-SNE analysis of equal data events in FlowJo.^{22 (link)}