Whole blood erythrocyte lysing kit

Immunodeficient BALB/c/RAG1 or RAG2^−/−γc^−/− mice were used to generate humanized mice (hu-mice). Hu-HSC mice were generated by injecting human fetal liver-derived CD34+ hematopoietic stem cells (HSC) intra-hepatically into newborn mice as we described previously (Berges et al., 2010 (link)). Mice were maintained at the CSU Painter Animal Center. These studies have been reviewed and approved by the Institutional Animal Care and Use Committee. Human fetal liver-derived CD34+ cells were purified and cultured for 24 hours in cytokine media (Akkina et al., 1994 (link); Hu et al., 2016 (link)). Neonatal mice were irradiated with 350 rads and injected intra-hepatically with 0.5–1×10⁶ human CD34 cells. BLT hu-mice were prepared by transplantation of fragments of human fetal liver and thymic tissues under the mouse kidney capsule as described previously followed by tail vein injection of autologous fetal CD34+ HSC (Akkina, 2013 (link); Hu et al., 2017 (link)). Transplanted mice were screened for human cell engraftment at 10–12 week post-reconstitution. Peripheral blood was collected and the red blood cells were lysed using the Whole Blood Erythrocyte Lysing Kit (R&D Systems, Minneapolis, MN). Fractioned white blood cells were stained with human CD4+5 FITC marker and FACS analyzed to confirm human cell engraftment (Berges et al., 2006 (link))

Humanized BALB/c Rag1^−/− or Rag2^−/−γc^−/− (Hu-HSC RAG-hu) mice were generated by engrafting human fetal liver-derived CD34+ hematopoietic progenitor cells (HPC) as previously described (Berges et al., 2008 (link); Berges et al., 2006 (link)). Mice were maintained at the Colorado State University Painter Animal Center and all studies were approved by the CSU Institutional Animal Care and Use Committee (Protocol 11-3153A). Newborn mice were preconditioned by irradiating with a sub lethal dose of 350 rads and then injected intrahepatically with 0.5–1 ×10⁶ human CD34+ cells. Mice were screened for human cell engraftment at 10–12 weeks post-reconstitution. Peripheral blood was collected by tail bleed and red blood cells were lysed by the Whole Blood Erythrocyte Lysing Kit (R&D Systems, Minneapolis, MN).The white blood cell fraction was stained against the human pan-leukocyte marker CD45 using hCD45-R-PE (Invitrogen) and FACS analyzed to determine the levels of human cell engraftment as previously reported (Berges et al., 2008 (link); Berges et al., 2006 (link)). Mice with more than 50% human cell engraftment were used for experiments involving hu-mice to assure robust numbers of human cells being present.

Human fetal, liver-derived, CD34 cells were isolated, column purified (Miltenyi Biotec, San Diego, CA) and cultured as previously described (Akkina et al., 1994 (link); Bai et al., 2000 (link); Veselinovic et al., 2016 (link)). CD34⁺ purity was assessed by flow cytometry. Neonatal Balb/c Rag1^−/−γc^−/− or Balb/c Rag2^−/−γc^−/− mice were preconditioned by irradiation at 350 rads and injected intrahepatically with 0.5–1 × 10⁶ human CD34⁺ cells per mouse (Berges et al., 2008 (link); Veselinovic et al., 2016 (link)). Transplanted mice were then screened at 10–12 weeks post-reconstitution for human cell engraftment. Peripheral blood was collected and the red blood cells were lysed using the Whole Blood Erythrocyte Lysing Kit and the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Fractioned white blood cells were stained with mouse anti-human CD45 FITC (eBioscience), CD3 PE (eBioscience) and CD4 PE/Cy5 (BD Pharmigen, San Jose, CA) for FACS analysis to confirm human cell engraftment (Berges et al., 2006 (link); Veselinovic et al., 2016 (link)). Mice were maintained at the Colorado State University Painter Animal Center. The studies conducted in this publication have been reviewed and approved by the CSU Institutional Animal Care and Use Committee.