u/ascorbicAcid1300

Hi I have a cell line infected with flu tagged to GFP for growth kinetics (24/48/72 hpi). Supernatant is harvested for quantification, and I want to harvest the cells for flow (GFP+) for the 3 timepoints. I have a vehicle (veh, negative) control in parallel.

Just wondering, when gating GFP+ cells at for each timepoint, should I gate it with the veh control of that particular timepoint? i.e. 24 h veh for 24 hpi; 48 h veh for 48 hpi; 72 h veh for 72 hpi. Or simply a random veh control suffices for all timepoints of my flu-GFP.

Hi I have a cell line infected with flu tagged to GFP for growth kinetics (24/48/72 hpi). Supernatant is harvested for quantification, and I want to harvest the cells for flow (GFP+) for the 3 timepoints. I have a vehicle (veh, negative) control in parallel.

Just wondering, when gating GFP+ cells at for each timepoint, should I gate it with the veh control of that particular timepoint? i.e. 24 h veh for 24 hpi; 48 h veh for 48 hpi; 72 h veh for 72 hpi. Or simply a random veh control suffices for all timepoints of my flu-GFP.

Hi I have 2 questions:

1. Currently I seed MDCK cells on 96 well plates one day before infection. Wondering whether I can seed the cells in serum-free medium directly, and proceed with infection on the same day (i.e. one day saved)?
2. In plaque assay we can have a "neat" dilution (i.e. undiluted) which lowers the limit of dilution (LOD). Can we do this for TCID50 too (with Reed-Muench method for analysis)? My current protocol is adding 35 ul of serially diluted virus onto 100 ul of seeded MDCK (first dilution is 0.5log). So for the first "dilution", can I directly undiluted 35 ul of virus instead?

Thanks!