Transplanted HILOs were harvested 26 days post transplantation and dissociated into single cells using TrypLE. The cells were stained by Zombie-UV staining Dye (BioLegend, 77474) to assess live vs. dead status. After blocking common epitope found in extracellular regions of mouse Fc-receptors by Fc block (Anti-mouse CD16/CD32 (Fc Shield) (70–0161-U500) staining, the antibodies (1:100 dilution) to the cell surface markers CD19 (PerCP/Cy5.5 anti-mouse CD19, BioLegend, 115533), Nk1.1 (anti-mouse Nk1.1 PE, eBioscience, 12–5941-81), CD45 (brilliant violet510 anti-mouse CD45, BioLegend, 103138), CD3 (brilliant violet650 anti-mouse CD3, BioLegend, 100229), Cd11b (anti-human/mouse APC-cyanine, TONBO, 25–0112U100) were used for FACS-based immune profiling. For flow cytometry analyses, data was collected using a BD Biosciences LSRII. For cell sorting, a BD Influx was used (100 micron nozzle tip and 1x PBS sheath fluid with sheath pressure set to 18.5PSI) with sample and collection cooling set to 4 degrees. Viable (Zombie-UV dye negative) single cells were selected for FACS or analyses using Forward scatter (FSC) and Side scatter (SSC) gating, followed by pulse-width discrimination for FSC and SSC.
Zombie uv staining dye
Manufactured by BioLegend
Zombie-UV staining Dye is a fluorescent dye used for the detection of dead cells in flow cytometry analysis. It binds to proteins in dead cells, emitting fluorescence upon ultraviolet light excitation. The dye enables the discrimination between live and dead cells in a sample.
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2 protocols using zombie uv staining dye
1
HILO Immune Cell Profiling Post-Transplant
2
HILO Immune Cell Profiling Post-Transplant
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Transplanted HILOs were harvested 26 days post transplantation and dissociated into single cells using TrypLE. The cells were stained by Zombie-UV staining Dye (BioLegend, 77474) to assess live vs. dead status. After blocking common epitope found in extracellular regions of mouse Fc-receptors by Fc block (Anti-mouse CD16/CD32 (Fc Shield) (70–0161-U500) staining, the antibodies (1:100 dilution) to the cell surface markers CD19 (PerCP/Cy5.5 anti-mouse CD19, BioLegend, 115533), Nk1.1 (anti-mouse Nk1.1 PE, eBioscience, 12–5941-81), CD45 (brilliant violet510 anti-mouse CD45, BioLegend, 103138), CD3 (brilliant violet650 anti-mouse CD3, BioLegend, 100229), Cd11b (anti-human/mouse APC-cyanine, TONBO, 25–0112U100) were used for FACS-based immune profiling. For flow cytometry analyses, data was collected using a BD Biosciences LSRII. For cell sorting, a BD Influx was used (100 micron nozzle tip and 1x PBS sheath fluid with sheath pressure set to 18.5PSI) with sample and collection cooling set to 4 degrees. Viable (Zombie-UV dye negative) single cells were selected for FACS or analyses using Forward scatter (FSC) and Side scatter (SSC) gating, followed by pulse-width discrimination for FSC and SSC.
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