The role of scavenger receptor BI in hepatitis - eTheses Repository ...

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4. After EP the cells were allowed to stand for 5 mins at RT prior to being
transferred into 10ml of pre-warmed DMEM + 10% FCS + P/S. 8ml of
the resuspended cells were placed in a T75 culture flask, and the
remainder put into 2 wells of a 24 well tissue culture plate to allow the
monitoring of HCV protein expression. The cells were immediately
taken into category 3 containment laboratories for culture and harvest
of particles.
5. At 48 hrs post EP, the efficiency of viral replication was quantified by
detection of the HCV non-structural protein NS5A. Briefly,
electroporated cells were stained using the immunofluorescence (IF)
protocol with saponin permeablisation (as detailed above), mouse anti-
NS5A mAb 9E10 was used at 1/200 dilution. JFH infected NS5A
positive Huh-7.5 cells are shown in Figure 2-2.
6. HCVcc particles were harvested between 4 and 14 days post EP, after
which the cells were discarded. To harvest, infected cells were cultured
in a minimal volume of DMEM + 3% FCS + P/S and media containing
secreted virions collected every 8-14 hrs. Harvested virus was frozen
prior to titration using the infection assay.