Stem cell factor (scf)

Human brain microvascular endothelial cells hCMEC/D3 (purchased from Meisen CTCC, Zhejiang, China) and microglial cells HCM3 (purchased from Meisen CTCC, Zhejiang, China) were cultured in DMEM medium (Gibco, Invitrogen, California, USA) containing 10% fetal bovine serum (FBS) (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin. Human mast cells LAD2 were cultured in RPMI 1640 medium (Gibco), containing 10% fatal FBS with 100 U/mL penicillin and 100 μg/mL streptomycin. For degranulation, LAD2 cells (purchased from Huzhen Company, Shanghai, China) were grown in StemPro-34 medium (Gibco) supplemented with 100 μg/ml stem cell factor (Novoprotein, Suzhou, China), 100 μg/ml IL-6 (Novoprotein), nutrient supplement (Gibco), 100 U/ml penicillin (Invitrogen, California, USA), 100 µg/ml of streptomycin (Invitrogen), and 2 mM L-Glutamine (Gibco).
HIV-NL4-3/spike pseudotyped virus was generated by Lipo 2000 transfection reagent (Invitrogen)-mediated co-transfection of HEK293T cells, with the spike-expressing plasmid pcDNA3.1-2019-nCoV-S-IRES (strain 2019-nCoV WIV04) and pNL4-3. Luc. ΔR ΔE. These two plasmids and HEK293T cells are provided by Dr. Lu Lu (Fudan University, Shanghai, China). Harvested HIV-NL4-3/spike pseudovirus were used to infect hCMEC/D3 and HMC3 cells for 48h, viral infection was measured by detecting luciferase activity (Promega, Madison, WI, USA).

For in vivo validation, plasmids encoding positive shRNA hits were modified to delete the puromycin resistance gene, amplified using an endotoxin-free DNA purification kit (12362, Qiagen), and packaged to generate retroviral supernatants as described above. For HPSC enrichment, wild type C57BL/6J mice were treated with 4 mg/mouse 5-fluorouracil (5-FU) (Sigma) by intravenous (iv) injection, followed by bone marrow cell collection 5 days later. HPSCs were stimulated with SCF (100ng/ml, Novoprotein), IL-3 (20ng/ml, Novoprotein) and IL-6 (25ng/ml, Novoprotein) for 36 hours before transduction. Retrovirus transduction of HPSCs was performed by spinoculation (2000rpm, 2 hours without brake) and media was replaced 4 hours later. Transduced cells were harvested after 20 hours for intravenous transfer into IgM^b-macroself mice. Alternatively, freshly isolated BM cells from mutant mice were depleted of red blood cells using ACK lysis buffer, counted, and resuspended in PBS. 1-10x10⁶ retrovirally transduced HPSCs or freshly isolated BM cells were iv injected into recipient mice. IgM^b-macroself mice were irradiated with 6 Gy by X-Ray before reconstitution. Recipient mice were euthanized and their splenocytes analyzed by flow cytometry 8 weeks after reconstitution.