Anti mouse ly 6a e sca 1 pe

Before the first 5-FU administration (day 65) and five days after every injection (days 75, 90 and 105), blood samples were extracted from the tail vein to analyze the effect of 5-FU administration on circulating hematopoietic stem cells. Blood samples were collected and prepared for immunostaining as previously described [7 (link)] and incubated for 30 minutes with following antibodies: anti-mouse Ly-6A/E (Sca-1) PE (12–5981 eBioscience), anti-mouse CD117 (c-Kit) APC (17–1171 eBioscience), mouse hematopoietic lineage eFluor 450 Cocktail (eBioscience 88–7772) and PE-Cy7 rat anti-mouse CD127 (560733 BD Biosciences). The number of hematopoietic stem cells (HSCs; lin-, Sca-1 +, c-kit +), common myeloid progenitors (CMPs; lin-, Sca-1 -, c-kit +), and common lymphoid progenitors (CLPs; Lin-, CD127+) were determined using a Gallios flow cytometer (Beckman Coulter) and the output was analyzed with Kaluza software (Beckman Coulter). The data were expressed as a percentage of the total selected events.

mHPCs were collected, washed with cold PBS containing 2% FBS and stained with fluorochrome conjugated monoclonal antibodies anti-mouse CD11b-APC (BD-Bioscience) or anti-mouse Ly-6A/E (Sca-1)-PE (eBioscience, San Diego, CA). Cells were acquired and analyzed on a FACS Canto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ) using the FACS Diva software (BD Biosciences, Bedford, MA).

The bone marrow was isolated from 4–6 weeks old donor mice (Coq9^+/+ or Coq9^R239X). Bone marrow cells were harvested from euthanized donor mice by CO₂ asphyxiation followed by cervical dislocation. The cells were flushed out of the 2 femurs and 2 tibias under sterile conditions, passed through a 40 μm cell strainer and treated with ammonium chloride solution (Stem Cell Technologies) to eliminate the red blood cells. Cells were stained with anti-mouse Ly-6A/E (Sca-1)-PE (eBioscience, San Diego, CA), washed with AutoMACS buffer and incubated with Anti-PE MicroBeads (Miltenyi Biotec) following the manufacturer´s instructions. An enriched mouse hematopoietic progenitor cells (HPCs) fraction was obtained after a magnetic separation using AutoMACS Pro Separator (Miltenyi Biotec). For transduction, 1x10⁶ mHPCs were incubated with LVs supernatant at MOI = 200 in Stem-Span Serum-Free Expansion Medium (StemCell Technologies) supplemented with 1% FBS, 1% penicillin/streptomycin and cytokines (mouse IL-3, murine SCF, human FMS-like tyrosine kinase 3 ligand [hFlt3L] and human IL-6) for 12 hours. After that, mHPCs were cultured in complete Stem-Span medium during 7 to 14 days.