Sfem medium

Lineage^−/low cells were isolated from WT BMCs using the lineage cell depletion kit (Miltenyi Biotech, BergischGladbach, Germany), in accordance with the manufacturer’s instructions. After isolation, lineage^−/low cells were seeded at a density of 5 × 10⁵ cells per well in low adsorption 24-well plates. Cells were cultured in SFEM medium (STEMCELL Technologies, Vancouver, Canada) supplemented with 10 ng/mL stem cell factor (R&D, USA), 10 ng/mL thrombopoietin (R&D, USA) and 10 ng/mL IL-3 (R&D, USA). During the study, the cells were exposed to either PBS or 15 mM IMP (MLBio, Shanghai, China) for 24 or 72 h, respectively. After harvesting, the cells were counted and stained with GMP surface markers before being subjected to analysis by FACS. For inhibitor experiments, MK2206 (MLBio, Shanghai, China) was added at a concentration of 5 mM for 1 h before the addition of IMP.
To evaluate the effects of AMP on myeloid lineage production, lineage^−/low cells were treatment with AMP (at 0, 50 μM, or 100 μM) for 72 h, and then changes in GMP frequency were assessed by FACS. In certain experiments, cells were treated with AMP or a control for 5 days. The cells were then harvested and stained with anti-mouse CD11b, anti-mouse F4/80, and anti-mouse Gr-1 for FACS analysis.

Production of lentiviral vectors was done according to Tiscornia, G^{53 (link)}: 3rd generation lentivector packaging plasmids for pRRL-Eμ-B29 vectors and 2nd generation lentivector packaging plasmids for LentiCRISPRv2GFP were co-transfected into 293T cells in the ratio of (15:10:5:4) (Lentivector:pMDL:pVSVG:pREV) and 2.8:2:1 (Lentivector pSPAX: pMD2G) respectively using ProFection Calcium Phosphate mammalian transfection system (Promega) or lipofectamine 30000 (Thermo) according to manufacturer’s protocol. Transfection medium was replaced 6–15 h after transfection with 5% serum DMEM serum and virus-containing supernatant was collected 24 and 48 h after replacement. The supernatant was then filtered with 0.45-μm PVDF filters (Millipore, MA, USA) and centrifuged in ultra-centrifuge using SW28 rotor for two and a half hours in 70,000 × g. The virus was reconstituted in 300–600 SFEM medium (STEMCELL Technologies, Vancouver, British Columbia, Canada). The concentrated virus was frozen at −80 °C until use. An aliquot of the frozen virus was used for titer in 018Z cells percentage of transduced cells was evaluated by flow cytometry using GFP, CRLF2, or IL7RA antibodies (Biolegend, CA, USA) Titer (infectious units/ml) was calculated according to Eq. (1):
Equation (1): titer calculation $\frac{\%transducedcellsX\#cellsatdayoftransduction}{total\mu lofvirus/well}X1000=virus\mathrm{IU}/ml$