Trimethoprim sulfa

For non-sorted transplants, the embryo equivalent used for each timepoint is as follows: ≥4 ee for e9.5, ≤3 ee for e10.5, and ≤1 ee for e11.5. For all transplants, single cell suspensions were resuspended in 50–70 μL FACS buffer for injection with defined numbers of adult helper BM added. For neonatal transplants, cells were injected into the facial vein of sublethally irradiated (180–200 Rads; XRAD 320, Precision X-ray) P1-P4 NSG recipients. Nursing NSG mothers were fed an antibiotic chow of Trimethoprim Sulfa (Uniprim, Envigo) for 4 weeks post transplant to prevent bacterial infections. For secondary transplantation into adult recipients, recipient C57BL/6 mice were conditioned with 800 Rads, anesthetized by isoflurane, and retro-orbitally injected with 1–2 million whole BM harvested from primary recipients. For peripheral blood analysis, blood was obtained from the tail vein of transplanted mice at various timepoints, and red blood cells were depleted using ACK lysis buffer. For BM analysis, BM was harvested from tibias and femurs by flushing with ice-cold FACS buffer followed by ACK lysis and filtration. Cells were stained with lineage antibodies and analyzed on the BD FACS-Aria II. FlowJo software (Tree Star) was used for data analysis.

Sorted cells were cultured for 16 h in 37°C and 5% CO₂. A well of either FLU‐treated or Vehicle‐treated TM5 cells was combined 1:1 with a well containing vehicle‐treated mTmG cells for a total of 2 × 10⁴ KLS donor sorted cells per recipient. These cells were transplanted via retro‐orbital injection into lethally‐irradiated isoflurane‐anesthetized gender matched C57BL/6 recipients. Lethal dose of X‐ray irradiation was 850 cGy single dose (XRAD 320, Precision X‐ray, North Branford, CT). Transplanted recipients were fed an antibiotic chow of Trimethoprim Sulfa (Uniprim, Envigo, East Millstone, NJ) for 3 weeks to prevent potential bacterial infections. For peripheral blood analysis, blood was obtained from the tail vein of transplanted mice at various time points, and red blood cells were depleted using ACK lysis buffer. For BM analysis, BM was harvested from tibias and femurs by flushing with ice‐cold FACS buffer (PBS with 2% fetal bovine serum) followed by ACK lysis and filtration. BM HSC population is defined as single cell, PI⁻, Ter119⁻, CD27⁺, c‐kit⁺, Sca1⁺, CD150⁺, cells with donor cells expressing CFP. Cells were analyzed on the BD Fortessa. Exclusion criteria include recipient mice without engraftment (chimerism below 1%), or recipients that died prior to analysis. FlowJo software (Tree Star) was used for data analysis.