u/ascorbicAcid1300

I transduced A549 lung cancer cell line with a lentivirus expressing a gene of interest. 48 h post-transduction, I infected the cells with flu for another 48 h before submitted to flow cytometry. Compared to the non lentivirus-transduced control, the %flu infected cells are significantly lowered, say reduced by 60-70%, which is not ideal.

Is the lentivirus competing with flu in terms of infecting the cells? If yes, does increasing the time between lentivirus and flu infection helps? Say when the lentivirus has been degraded, then I infect the cells with flu, there shouldn't be any competition.

Or are there any other factors? Thanks!

Hi, has anyone tried adding cell suspension (with trypsin and DMEM w/ 10% FBS) into BD Cytofix/Cytoperm at a 1:1 (v/v) ratio? Will any components significantly impact the fixation/permeabilization efficiency of the solution for flow cytometry of intracellular proteins?

I have been doing a PBS washing step, but later I will be doing an experiment involving a few hundred samples, so I am thinking of shortcuts. Thanks!

Hi, has anyone tried adding cell suspension (with trypsin and DMEM w/ 10% FBS) into BD Cytofix/Cytoperm at a 1:1 (v/v) ratio? Will any components significantly impact the fixation/permeabilization efficiency of the solution for flow cytometry of intracellular proteins?

Hi I am copying Prism figures (through the "copy graph" function) and pasting to Adobe Illustrator. But the axis always passes through during the X-Y axis intersection (not seen in Prism) when pasted in AI.

https://preview.redd.it/0mlzs5nxpleh1.png?width=605&format=png&auto=webp&s=578ba1e494aaec6db5cb6c7eda8c3e3f7088160a

How to solve this issue? Thanks!

Hi I am copying Prism figures (through the "copy graph" function) and pasting to Adobe Illustrator. But the axis always passes through during the X-Y axis intersection (not seen in Prism) when pasted in AI.

https://preview.redd.it/y5mibvznpleh1.png?width=229&format=png&auto=webp&s=b1a4a4843ed21b47a1fb90d9bb927ec44f7ae6b1

How to solve this issue? Thanks!

A FBS-free medium needs to be used for incubating cells infected with most IAV, as FBS inhibits TPCK-trypsin. It's completely fine for MDCK cells, but I am tasked with more delicate cells like A549, which, from my last few trials, are very unhappy without FBS (stressed and floated - even for mock infection control so should not be CPE).

I saw from the internet that we can add ~0.25%(w/v) BSA to the infection medium instead, but not sure what's the best concentration range. More importantly, how to make TC-grade BSA solution? We can't filter BSA, nor can we autoclave it.

Hi all I am somehow stuck with infecting PR8 on A549 cells for simple growth kinetics assay (collect at 1, 24 48, 72 hpi). Here are my conditions:

Infecting PR8 at MOI 0.01 on A549 in 24 well plates

Wash with PBS x 1

Inoculate virus in 300 uL DMEM w/o FBS for 1 h in 37 C incubator

Wash with PBS x 1

Incubate cells with 500 uL DMEM w/o FBS, w/ 0.5 ug/mL TPCK-trypsin

Readout: TCID50 assay

I have used this virus to infect MDCK before and it's fine. Just not sure what went wrong as PR8 should be a robust IAV in my lab.

Thanks!

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!