Easysep release mouse pe positive selection kit

For adoptive transfer of BM MKs, GFP mice (donor) and Mpl^hlb219 mice (recipient) were simultaneously exposed to a single dose of 5 Gy TBI. Then, 3 × 10⁵ BM MKs from GFP mice, Igf1^f/f or Igf1^ΔMK mice at 2 dpi were obtained by continuously enriching CD41⁺ cells and CD42d⁺ cells using an EasySep™ Release Mouse Biotin Positive Selection Kit and an EasySep™ Release Mouse PE Positive Selection Kit (all StemCell Technologies, Vancouver, BC, Canada) with biotin-labeled anti-CD41 and PE-labeled anti-CD42d antibodies according to the manufacturer’s instructions. Subsequently, the freshly-isolated MKs were intravenously transfused into Mpl^hlb219 mice.

CD45.1⁺ γδ T cells were obtained from the spleens and peripheral lymph nodes according to the protocol provided by the EasySep™ Release Mouse PE Positive Selection Kit (StemCell Technologies Inc., Canada) and cocultured with CD45.2⁺ DNT (1:1) in a round-bottom 96-well plate for 24 h. Annexin V and caspase-3 staining were performed to detect the apoptosis of CD45.1-positive cells via flow cytometry.
Regarding the anti-NKG2D blocking assay, CD45.2⁺ DNT were preincubated with an anti-NKG2D antibody for 1 h before being cocultured with CD45.1⁺ γδ T cells. After 24 h of coculture, Annexin V and caspase-3 staining were performed to detect the apoptosis of CD45.1-positive cells via flow cytometry.