Primary human dermal LEC isolated from adult skin and human aortic endothelial cells (HAoEC) were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured in endothelial cell growth medium MV 2 (PromoCell) containing 5% heat-inactivated fetal bovine serum (FBS), 100 IU/mL of penicillin, 100 μg/mL streptomycin, and growth factors bullet kit. Cells were maintained in a humidified incubator at 37°C and used till passage 7. Primary mouse lung LEC were isolated after digestion of lungs with collagenase type 2 (1 mg/mL) for 1 h at 37°C as we described previously 24 (link). LYVE-1-positive cells were separated and cultured.
A smart pool of siRNA for human CD47 (M-019505-01-0005) and a non-targeting control siRNA (D-001210-01-05) were purchased from Horizon Discovery (Cambridge, United Kingdom). Cells were transfected with siRNAs using the TransIT-TKO transfection reagent (Mirus Bio LLC, Madison, WI, USA) according to the manufacturer’s instructions. Cells were used for further experiments 48 h post-transfection and gene silencing was confirmed using qRT-PCR. Protein knockdown was validated by Western blot experiments.
A smart pool of siRNA for human CD47 (M-019505-01-0005) and a non-targeting control siRNA (D-001210-01-05) were purchased from Horizon Discovery (Cambridge, United Kingdom). Cells were transfected with siRNAs using the TransIT-TKO transfection reagent (Mirus Bio LLC, Madison, WI, USA) according to the manufacturer’s instructions. Cells were used for further experiments 48 h post-transfection and gene silencing was confirmed using qRT-PCR. Protein knockdown was validated by Western blot experiments.