Streptavidin alexa647

The following antibodies (BioLegend) were used for flow cytometry: Biotin-conjugated anti-mouse Sca1 (D7), BV421-labeled anti-mouse Sca1 (D7), APC-labeled anti-mouse EpCAM (G8.8), FITC-labeled anti-mouse EpCAM (G8.8), PE-labeled anti-mouse CD24 (M1/69), Biotin-conjugated anti-mouse CD24 (M1/69), Biotin-conjugated anti-mouse CD81 (Eat2), Streptavidin-APC, and Streptavidin-PECy7. For immunofluorescence staining of sections, the following antibodies were used: Biotin-conjugated anti-mouse CD81 (Eat2, BioLegend), PE-labeled anti-mouse CD81 (Eat2, BioLegend), Purified anti-mouse Sca1 (e13-161.7, BioLegend), Biotin-conjugated anti-mouse Sca1 (177228, R&D Systems), Streptavidin-Alexa647 (BioLegend), Alexa488-labeld anti-Rat IgG (Jackson). Dead cells were excluded by propidium iodide (Sigma) or Zombi NIR (BioLegend) staining.

Ca²⁺ mobilization in YC3.60-expressing cells was analyzed by flow cytometry using CyAn ADP™ (Beckman Coulter) equipped with a 405-nm, solid-state laser as previously described. At 405-nm excitation, FRET intensity was calculated as the ratio of YFP to CFP intensity. Cell sorting was performed using a MoFlo XPD cell sorter (Beckman Coulter) in the Stem Cell Laboratory at our university. Antibodies with the following specificities were conjugated in-house: anti-CD19-Alexa647 and ant-B220-Alexa647. Streptavidin-PE and streptavidin-Alexa647 (which were obtained from BioLegend) were used as secondary reagents. Anti-CD3-PE, anti-CD4-PE, anti-GL-7-APC, anti-B220-Pacific Blue, anti-IgM-PE-Cy7 and anti-CD138-PE were also obtained from BioLegend.