Hello everyone. I need to use Promega Dual Glo assay for HEK293 cells, but I am really afraid of wash out my cells from the 96-well plate, and at the same time, I am afraid to use coating because I have heard that using coating may decrease the transfection level.
So, I have several questions:
Thanks!
You could try to use the "Reverse Transfection". I seeded 75uL of 1x10^4 HEK293ft cells (from a 70-90% confluence Petri dish) together with 10uL Lipocomplex (TransIT-X + 100ng of plasmid).
Firstly I added the Lipocomplex in the middle of the 96well, thereafter I mixed them with the 75uL HEK293 cells (resuspend it and distribute well around the well).
The first-second day the cellular division is slowly (because of the Lipocomplex) , but later the division efficiency will increase .
Adrián Mallén, thanks a lot. I am already using reverse transfection. The problem is that I have only Lipofectamine 2000 now, and as far as I understand I need to wash my cells after it because it is toxic to them. I am going to change the reagent, but also I need to test some things ASAP, that is why I am trying to find some other way. Unfortunately, I completely forgot to put this thing in my question. Anyway, thank you, I will try to use your protocol further.
Why don't you consider higher cell concentration? Transfection in bigger plate means higher volume of reagents.
Hi Yulia, I used the Lipofectamine2000 in previous experiments and it was the same. I didn't need to wash my cells because they were harvested on day 3:
1. I seeded HEK293+Lipocomplex on day 1 in a standard 96well plate.*You can use a control well transfecting GFP plasmid (so you can evaluate the transfection efficiency the next day in the microscope).
2. I harvested HEK293 cells 72h after (day 3) by adding the 1st Firefly reagent (which also contains lysis buffer) and resuspending.
3. Immediately, transfer the hek293cells+firefly reagent to an OPAQUE 96 PLATE. Wait 30min. Perform the 1st luciferase assay.
4. Add the 2nd Renilla reagent. Wait 30min (the same as with the 1st reagent) and perform the 2nd luciferase Assay.
Thanks a lot, Adrian.
I have never tried to leave my cells with Lipofectamine2000, but I will try as you recommend it.
Also, I measure my assay results in a growing plate, but I am fine with it because the background is always very low, even though I use clear microplates. I do not know anything about your experiment, but maybe it helps you reduce costs. My background level is always about 40 CPS with thousands of CPS for experimental wells.
Mukesh Kumar Gupta, thank you for your answer.
I am afraid both to overgrow and to wash out my cells. Unfortunately, I am not familiar with cell culturing yet, my previous work was in a completely different field.
What amount of cells per well would you recommend?
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