Facs sortware

To obtain HEK-293T cells with stable expression of ACE2 protein, a lentiviral system bearing ACE2 (Genbank ID: BAJ21180.1) gene was constructed. In brief, HEK-293T cells (ATCC) with 70%–80% confluence in a 10 cm dish were co-transfected with 12 μg of plasmid pHIV-puro encoding RRE and ACE2 genes, 8 μg of plasmid psPAX2 encoding gag and pol, and 4 μg of plasmid VSV-G encoding G glycoprotein of vesicular stomatitis virus(VSVG) using Lipofectamine 3000 Reagent (Invitrogen). Twelve hours later, the medium was changed to fresh DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) for another 48 h culturing. Medium containing virus particles was harvested and concentrated using a Lentivirus Concentration Kit (Genomeditech). The concentrated virus particles were used to infect HEK-293T cells under selection pressure of 10 μg/mL puromycin (Beyotime Biotechnology). The transfection efficiency was examined by flow cytometry using S1-mFc recombinant protein (Sino Biological) as primary antibody and FITC-AffiniPure Goat Anti-Mouse IgG (Jackson) as secondary antibody. The resulting bulk transfected population was sorted on a BD FACSJazz Cell Sorter (BD) with the BD FACS™ Sortware. Cells with top 1% fluorescence intensity were retained and expanded for subsequent use.

Samples were run and 1,000,000-10,000,000 events were collected using either BD FACS Diva or BD FACS Sortware with a BD Aria II or BD Influx cell sorter respectively (BD Biosciences, Franklin Lakes, NJ). The BD Aria II was equipped with a 50 mW 405 nm laser, 20 mW 488 nm laser, and 18 mW 633 nm laser while the BD Influx was equipped with a 100 mW 355 nm laser, 200 mW 488 nm laser, and 120 mW 640 nm laser. Hoechst 33342 was excited using the 355 nm laser and detected through a 460/50 filter. Hoechst 34580 was excited using the 405 nm laser and detected through a 460/50 filter. JC-1, anti-CD71 PerCP eFluor 710 and Streptavidin PE-Cy7 were excited using the 488 nm laser and detected through 530/40, 692/40, and 750LP filters respectively. Atto633 and anti-CD45 APC eFluor 780 were excited using the 633 nm laser and detected through 670/30, 750LP filters respectively. The RBC and leukocyte population was selected based on FSC/SSC properties and single cells were gated based on either trigger pulse width or by using the FSC peak area to height ratio. Cell sorting was performed on the BD Influx into collection tubes and slides prepared as described earlier. Compensation and further analysis was performed using FlowJo v10.0.6 (Tree Star, Ashland, Oregon, USA).

Culture samples were diluted 1:1,000 in 500 μl of PBS. Fluorescence intensity of NeonGreen and mCherry in cells was monitored by flow cytometry analysis on a BD FACSJazz (BD Biosciences), and data were acquired with the BD FACS Sortware. mNeonGreen and mCherry were excited with 488 and 561 nm solid-state lasers, and their emission was detected using 513/17 and 610/20 nm emission filters, respectively. For each sample, fluorescence of 20,000 cells was captured, and the data was analyzed using FCS Express 7 (De Novo Software).