Lsrii multi laser analyzer

To assess the susceptibility of hNTCP-eGFP HepG2 clones to HBV infection, cells that had been challenged with HBV were first trypsinized and then fixed with FACS fixation buffer (1% PBS, 1% PFA) for 20 min at RT. After fixation, cells were centrifuged and re-suspended in permeabilization buffer (1% FBS, 0.1% saponin, in 1× PBS) for 20 min at RT. After permeabilization, cells were again centrifuged and were re-suspended with 50 ul of HBcAg primary antibody (1:200 diluted in permeabilization buffer; HBcAg goat-anti-mouse (Fisher Scientific, cat# MA7609 Waltham, MA) and incubated for 30 min at 4 °C. After the incubation cells were washed twice with FACS Buffer (1× PBS, 1% FBS). Cells were then re-suspended and incubated with an Alexa 647 anti-mouse secondary antibody (1:250 dilution in permeabilization buffer; Fisher Scientific, Waltham, MA) for 30 min at 4 °C. Cells were then washed twice with FACS buffer to remove any excess secondary antibody and were then run on a LSRII Multi-Laser Analyzer (BD, Franklin Lakes, NJ) at the Princeton flow cytometry core facility.

Lentiviral pseudo-particles were generated by co-transfecting 4.0E + 6 293T cells in a 10 cm tissue-culture plate using Xtremegene (Sigma-Aldrich, St. Louis, MO) with plasmids expressing the respective pLVX-NTCP-tagRFP proviral DNA, HIV-1 gag–pol, and VSV-G at a ratio of 1/0.8/0.2. Supernatants were collected at 24, 48 and 72 h, pooled, and filtered through a 0.45 μm filter (Millipore, Burlington, MA). Filtered lentiviral supernatants were supplemented with polybrene (4 μg/mL, vol/vol) and (1:50, vol/vol) 1 M HEPES, aliquoted, and stored at −80 °C until use. Next, HepG2 cells were transduced with the human-, OWM- (with or without humanizing mutations), NWM- (with or without humanizing mutations), or prosimian-NTCP-tagRFP lentiviruses. After 3 days, expression of the fusion protein was assessed by both fluorescence microscopy using an EVOS microscope (Fisher Scientific, Waltham, MA) and LSRII Multi-Laser Analyzer (BD, Franklin Lakes, NJ) at the Princeton flow cytometry core facility. Each cell line generated showed greater than 90% of the cells expressing the respective fusion construct.