Id7000 spectral cell analyzer flow cytometer

For neutralization assays, A549 cells were seeded in 96-well plates at 50,000 cells/well. At 6 h after plating, the SFV viruses were incubated for 30 min with or without heat-inactivated (56 °C for 30 min to inactivate the complement system) rhesus macaque sera before being added to the cells. The cells were harvested 48 h post-infection by brief trypsinization, followed by the addition of one volume of 5% FCS in PBS to inhibit trypsin activity and fixed by addition of one volume of PBS supplemented with 4% formaldehyde (Carl Roth). Ten thousand cells were analyzed per sample for YFP expression on an ID7000™ Spectral Cell Analyzer flow cytometer (Sony Biotechnology, San Jose, CA, USA). Data were analyzed using ID7000™ Spectral Cell Analyzer (Sony Biotechnology). Two independent experiments were performed for both SFVs. Each experiment was normalized to cells without immune sera infected with the respective virus.

For infection assays, cells were plated at 50,000 cells/cm² (SLK, 293T). At 6 h after plating, the KSHV virus was incubated for 30 min with or without heat-inactivated mouse sera before being added to the cells. The cells were harvested 48 h post-infection by brief trypsinization, followed by the addition of one volume 5% FCS in PBS to inhibit trypsin activity and fixed in PBS supplemented with 4% formaldehyde (Carl Roth). 10,000 cells were analyzed per sample for GFP expression on an ID7000™ Spectral Cell Analyzer flow cytometer (Sony Biotechnology, San Jose, CA, USA). Data was analyzed using ID7000™ Spectral Cell Analyzer (Sony Biotechnology). Three independent experiments were performed for KSHV and KSHV gH-ASAELAAN. Each experiment was normalized to SLK cells without immune sera infected with the respective virus, and afterwards, averaged for each mouse sera.