Streptavidin conjugated nanobeads

For magnetic isolation of blood neutrophils, red blood cells were lysed as above, and single cell suspensions were washed in selection media. Cells were then incubated with Fc block for 10 min on ice as noted above. Neutrophils were isolated using the MojoSort Mouse Ly6G Selection Kit (Biolegend, Cat# 480,058) according to manufacturer’s instructions. For magnetic isolation of femur bone marrow LSK + cells, single cell suspensions were blocked and stained with biotinylated-lineage cocktail (Biolegend, Cat# 133,307) and stem/progenitor cells were negatively selected using streptavidin-conjugated nanobeads (Biolegend, Cat# 480,015). Lineage-negative cells were then stained with c-Kit-APC (Biolegend, Cat# 105,811) for positive selection with anti-APC-conjugated nanobeads (Biolegend, Cat# 480,071). Finally, Lin-cKit + bone marrow cells were stained with Sca1-APC (Biolegend, Cat# 108,111) for positive selection with anti-APC-conjugated nanobeads. The resulting neutrophil and LSK + cell populations were stored in Qiazol lysis reagent (Qiagen, Cat# 79,306) at -80 °C.

For magnetic isolation of blood neutrophils, red blood cells were lysed as above, and single cell suspensions were washed in selection media. Cells were then incubated with Fc block for 10 min on ice as noted above. Neutrophils were isolated using the MojoSort Mouse Ly6G Selection Kit (Biolegend, Cat# 480058) according to manufacturer’s instructions. For magnetic isolation of femur bone marrow LSK+ cells, single cell suspensions were blocked and stained with biotinylated-lineage cocktail (Biolegend, Cat# 133307) and stem/progenitor cells were negatively selected using streptavidin-conjugated nanobeads (Biolegend, Cat# 480015). Lineage-negative cells were then stained with c-Kit-APC (Biolegend, Cat# 105811) for positive selection with anti-APC-conjugated nanobeads (Biolegend, Cat# 480071). Finally, Lin-cKit+ bone marrow cells were stained with Sca1-APC (Biolegend, Cat# 108111) for positive selection with anti-APC-conjugated nanobeads. The resulting neutrophil and LSK+ cell populations were stored in Qiazol lysis reagent (Qiagen, Cat# 79306) at −80 °C.