Anti c kit igg allophycocyanin apc secondary antibody

CSCs were isolated[48 (link)] and purified[3 (link)] using previously published methods with some modifications. The rats were deeply anesthetized with sevoflurane, and the atrial appendage was sliced and digested with 0.1% collagenase type II (Sigma, USA). After a 40-min digestion at 37°C, the cells were collected by sedimentation at 1200 rpm for 5 min. Then, the cells from the atrial appendage were incubated in a humidified chamber in Ham’s F12 medium containing 10% fetal bovine serum (FBS), 1% penicillin and streptomycin, 1% L-glutamine, 20 ng/ml human recombinant fibroblast growth factor, 20 ng/ml leukocyte inhibitory factor, and 10 ng/ml epidermal growth factor (EGF). After reaching >90% confluence, the cells were resuspended by trypsinization. Subsequently, the CSCs were incubated with a rabbit anti-C-kit antibody (1:250 in F12 medium) for 1 h and sorted with anti-rabbit secondary antibody-conjugated 2.8 μm magnetic beads (Dynal Biotech, Norway) for 30 min as instructed by the manufacturer's protocol. The purified C-kit⁺ CSCs were cultured in the previously mentioned F12 medium. Flow cytometry (FCM) was performed to confirm the surface markers on the C-kit⁺ CSCs. The cells were incubated with the fluorochrome-conjugated anti-CD34-PE, anti-CD45-PE, and anti-C-kit primary antibodies and the anti-C-kit IgG-allophycocyanin (APC) secondary antibody (all from BioLegend, USA).

CSCs were isolated [58 (link)] and purified [59 (link)] using previously published methods, with some modifications. Rats were deeply anesthetized with sevoflurane, and the atrial appendage was sliced and digested with 0.1% collagenase type II (Sigma, USA). After about 40 min digestion at 37°C, cells were collected by sedimentation at 1200 rpm for 5 minutes (min). Then, cells from the atrial appendage were incubated in a humidity chamber in Ham's F12 medium containing 10% FBS, 1% penicillin and streptomycin, 1% L-glutamine, 20 ng/mL human recombinant fibroblast growth factor, 20 ng/mL leukocyte inhibitory factor, and 10 ng/mL epidermal growth factor (EGF). When cells confluence reached >90%, they were suspended by trypsinization. Then, CSCs were incubated with rabbit anti-c-kit antibody (1 : 250 in F12 medium) for 1 hour (h) and sorted out with anti-rabbit secondary antibody conjugated 2.8 μm magnetic beads (Dynal Biotech, Norway) in 30 min as instructed by the manufacturer's protocols. The purified c-kit⁺ CSCs were cultured in the aforementioned F12 medium. Flow cytometry was used to confirm the expression patterns of CSCs markers. Cells were incubated with fluorochrome-conjugated primary antibodies: anti-CD34-PE, anti-CD45-PE, and anti-c-kit primary antibody and anti-c-kit IgG-allophycocyanin (APC) secondary antibody (all from BioLegend, USA).