hello,

I am doing BrdU to compare proliferation after transfection in HPDLF cells by using BrdU Roche Kit III.

There are three groups (a)control (b)empty plasmid (c) experimental E#plasmid

There are 6 replicates in each group. The number of cells is 7500/100uL cells per well.

I would like to account for 12 hr, 24 hr, 36 hr and 48 hr after transfection.

My question is

(1) when shall I add BrdU labelling solution after transfection?

for example, for 12hr, BrdU labelling is added after 12 hr of transfection,

for 24 hr, BrdU labelling is added after 24 hr of transfection and same for 36 and 48 hr. Then every 4 hr, fixation and detection of result?

OR

BrdU labelling agent is added since first in every plate

(12hr, 24hr, 36hr, 48hr) after 12 hr of transfection.

Then detection after 12 hr, after 24 hr, after 36 hr and after 48 hr?

(2)How to seed the cells and make the groups for analysis?

eg A1to A6 -plate blank( no media)

B1 to B6 -Blank Group (only media no cells)

C1to C6 ( Control)

D1 to D6 (Empty plasmid)

E1 to E 6 ( Experimental Plasmid)

is that correct?

(3)Do we need a standard?

(4) how to analyse the data?

the protocol said

"Measure the extinction of the samples in a microplate reader at 405 nm with a reference wavelength at approx. 490 nm (e.g., EAR 340 ATTC, SLT Lab instruments)"

it means we can measure the absorbance at the reference at 405 or 490? or need to set 2 wavelengths?

Thank you very much for your kind answers