All cell culture steps were carried out in an incubator with 5% CO2 at the indicated temperatures. On day 0, Huh7.5 or Huh7.5ΔACAT cells were set up in medium F at a density of 8x104 cells per well of a 24-well plate. On day 1, the media was removed and replaced with 1 ml of medium F supplemented with 5 µM of the indicated oxysterol. After 4 hr at 37 °C, the media was removed and replaced with 200 µl of either hCoV-OC43 virus or ZIKV-MR766-Venus virus in OptiMEM and incubated at 33 °C or 37 °C, respectively. After 1 hr, 800 µl of medium F was added to each well and maintained at either 33 °C or 37 °C as indicated above. After 24 hr, cells were harvested with Accumax, mixed with a 4% (v/v) stock solution of PFA in PBS (1% final concentration), and incubated for 10 min at RT, following which cells were analyzed by FACS either immediately or stored at 4 °C and processed at a later time.
Zika-infected cells were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatants were removed, and the cell pellets were resuspended in 150 µl of buffer E prior to FACS analysis.
OC43-infected cells were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatants were removed, and the cell pellets were resuspended in 50 µl of BD Cytofix/Cytoperm Solution. After incubation for 20 min at RT, 150 µl of BD Wash/Perm Solution was added to each sample, after which samples were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatant was removed, and cell pellets were resuspended in 200 µl of BD Wash/Perm Solution, and subjected to centrifugation at 1500 x g for 5 min at RT. After removing the supernatant, cell pellets were resuspended in 50 µl of anti-coronavirus group antigen antibody (1:50). After incubation for 30 min at RT, 150 µl of BD Wash/Perm Solution was added, and samples were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatant was removed, and cell pellets were resuspended in 200 µl of BD Wash/Perm Solution and subjected to centrifugation at 1500 x g for 5 min at RT. The supernatant was removed, and cell pellets were resuspended in 50 µl of Goat anti-mouse AlexaFluor488 (1:1000). After incubation in the dark (wrapped in aluminum foil) for 30 min at RT, 150 µl of BD Wash/Perm Solution was added and samples were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatant was removed, and cells were resuspended in 200 µl of BD Wash/Perm Solution and subjected to centrifugation at 1500 x g for 5 min at RT. After this final step, the supernatant was removed, and cells were resuspended in 150 µl of buffer E and analyzed on a Stratedigm S1000 EX Flow Cytometer using the GFP channel. Infection levels were determined as the percentage of Venus-positive cells (indicating Zika infection) or AlexaFluor488-positive cells (indicating OC43 infection) from at least 7500 single cells per replicate, as assessed by FlowJo software.
Zika-infected cells were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatants were removed, and the cell pellets were resuspended in 150 µl of buffer E prior to FACS analysis.
OC43-infected cells were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatants were removed, and the cell pellets were resuspended in 50 µl of BD Cytofix/Cytoperm Solution. After incubation for 20 min at RT, 150 µl of BD Wash/Perm Solution was added to each sample, after which samples were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatant was removed, and cell pellets were resuspended in 200 µl of BD Wash/Perm Solution, and subjected to centrifugation at 1500 x g for 5 min at RT. After removing the supernatant, cell pellets were resuspended in 50 µl of anti-coronavirus group antigen antibody (1:50). After incubation for 30 min at RT, 150 µl of BD Wash/Perm Solution was added, and samples were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatant was removed, and cell pellets were resuspended in 200 µl of BD Wash/Perm Solution and subjected to centrifugation at 1500 x g for 5 min at RT. The supernatant was removed, and cell pellets were resuspended in 50 µl of Goat anti-mouse AlexaFluor488 (1:1000). After incubation in the dark (wrapped in aluminum foil) for 30 min at RT, 150 µl of BD Wash/Perm Solution was added and samples were subjected to centrifugation at 1500 x g for 5 min at RT. The supernatant was removed, and cells were resuspended in 200 µl of BD Wash/Perm Solution and subjected to centrifugation at 1500 x g for 5 min at RT. After this final step, the supernatant was removed, and cells were resuspended in 150 µl of buffer E and analyzed on a Stratedigm S1000 EX Flow Cytometer using the GFP channel. Infection levels were determined as the percentage of Venus-positive cells (indicating Zika infection) or AlexaFluor488-positive cells (indicating OC43 infection) from at least 7500 single cells per replicate, as assessed by FlowJo software.
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