Pe anti his tag antibody

Flow cytometry was conducted as previously described [29 (link),31 (link)]. Briefly, each of the ACE2-encoding plasmids was transfected into HEK293T cells at a confluency of 80% in 6-well plates using lipofectamine3000 reagent (Invitrogen). After 48 hours, the cells were detached using Accutase (Millipore), incubated with Human TruStain FcX (BioLegend) for 10 minutes on ice, and dissolved in the flow cytometry buffer containing 5% fetal bovine serum (Invitrogen) and 1mM EDTA (VWR) at numbers of approximately 10⁶ cells. To measure the level of SARS-CoV-2 RBD attaching to the cell surface, ACE2-transfected HEK293T cells were first incubated with 2 μg of purified His-tagged RBD on ice for 1 hour, stained with rhodopsin antibody Alexa Fluor 647 (Santa Cruz Biotechnology) at 1:200 dilution and 5 μl PE anti-His tag antibody (BioLegend) on ice for 20 minutes, and finally resuspended in the flow cytometry buffer. Fluorescent signals on the cellular surface were immediately analyzed using Symphony A3 (BD Biosciences), FlowJo software (Ashland) and GraphPad Prism version 9.5.0 with one-way analysis of variance (ANOVA). All measurements were independently repeated at least for biological triplicates.

Briefly, cells were harvested with saline sodium citrate buffer pH 8.0 and incubated with 5% sheep serum for 30 min42 (link),44 (link). Cells were subsequently incubatedwithPE-anti-rhPLAC1 Ab (5 μg/ml), PE-anti-his tag antibody (Biolegend, San Diego, CA, USA) (dilution: 1:100) or PE-isotype control (5 μg/ml) (Sina Biotech) for one hr. For intracellular PLAC1 staining, cells were fixed using 1.5% formaldehyde for 15 min. Cells were then permeabilized using 0.5% saponin for 15 min, incubated with 5% sheep serum for 30 min followed by PE-anti-rhPLAC1 Ab (5 μg/ml) incubation for one hr. Cells were analyzed by a flow-cytometer (Partec, Munster, Germany).