HI,

in my recent past, i have worked with these cell lines for transfection. what do you want to know exactly

thanks

Ok, sir i have problem with HEK293 cell line culturing, upto P3 the cell attache and confluent upto 70 to 80 percent,but when i freez one flask of that cell line, and after about three months, i review that vial so much cell and live but not attached and float and very little cell atttach weekly, so what will be reson for that problem. Regard Muhammad waseem(ICCBS,Pakistan)

how do you freeze the cells.

while freezing the medium should have 10% of DMSO and frozen at a conc of 1-2 million cells/ml.

after thawing there is loss of cells. But the cells which are attached to the flask, will grow with the change of medium intermittently.

thanks

by the way, how do you freeze your cells

Trypsinise the cells, then resusupent the pellet with 10%FBS complet medium .5ml and then add 5%DMSO .5ml, and keep it firstly for one hour in -20 and then for 24hr in -80 next day transfer into Liquid nitrogen.

In start they are growing, adher and confluent normall but after P3, that problem come.Now what i do to for that.N thank,s for yr reply.

the cells loose adherence after P3 or these cells after freezing, when thawed are not very adherent

May be that will be the reson that cells after P3 attaching property becom low.

BY the thank,s alot.

But one question i will ask from u is that, that what concebtration of plasmid can be use for transfection.

concentration of the plasmid depends on the experiment. If your gene is tagged with some fluorescent protein like GFP or RFP, you can visualise the looks under the microscope for the transfection efficiency. Generally you can start with 0.5,1, 2 ug of the plasmid, look for the transfection efficiency. If the transfection efficiency does not increase wih the plasmid concentration, you can use the lowest concentration of the plasmid.

Be careful while using the LIpofectamine (or any reagent).do not pipete the lipofectamine and DNA mix too much, just incubate in hood for 5 minutes, and then add drop by drop on the cells.

thanks

OK,but sir i use strategen kit for transfection and plasmid concebtration rang is 10-30ug and my plasmid tagged with GFP.Thank,s.

as far as my knowledge goes, 10ug is too high. what is the transfection reagent in the kit?

any ways, as recommended you can start with 10ug, but also at 5 and 2 ug of DNA, and check for GFP expression in fluorescence microscope and count the GFP Positive cells in all the three cases.

I will use Capo4 method for transfection.

Dear,when transfect the HEK293 cells after 2 to 3 hr cell lyse,and then centrifug that cells in supernaten will be Adenoviral vector,so i want to know that,that can store that at -80.or will add directly into Msc.

sorry, i did not understand

Dear, that within trasfection HEK293 cell lyse(Adenoviral vector production)then i will centrifug that cells in supernatant their will be viral particle,so now can we store that Adenoviral vector at -80c,or otherwise will be use directly.

if i understand correctly, after transfection, the cells lyse. the virus is released in to the supernatant, which can be obtained by centrifuging the cells.

so, now the supernatnat contains the virus. If you want the virus to be used again immediately, it can be used for another round of transfection. If there is no immediate use, you can store the virus at -80, or liquid nitrogen for long term storage or at 4 degress for short term storage

Ok, sir i want that.And what will be the condition for (means will be stored in glycerol?) and the other question is that.Plasmid dilution is in deionized water, but i have plasmid in TE buffer.

By the way many many thank,s for your kind responce.

no need of any agent. you can directly store at the required temperature.

plasmid can be diluted with water, though it is in TE buffer.

One small suggestion, if survival of cell is problem. try Corning Cell Bind surface for revival of cells from frozen vials, you will get better attachment

Thank you so much. I posted my question i think 7 years ago. That time i just started my PhD project.

Any how thank you so much.