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Transfecting primary cells in suspension - (Aug/26/2007 )

Hi friends, I am trying to transfect the human primary epithelial cells which are grown in suspension. Till now the stable transfection using retro & lenti has worked quite good. But we need to do transient transfections in the same cells for some experiments. I have tried with Lipofectamine-2000 & Fugene-6 but did not see ANY transfection!(GFP reporter). Is there any special things to remember for primary cell transfection?(when u dont want to use virus!). Thanks for any suggestion.


What type of epithelial cells are these?
Are these growing in the form of individual cells or as spheroids/clusters?
Were you be able to transfect them when they are adherent?
I had some success with suspension transfection of bovine endothelail cells using lipofectamine 2000.
I did it with trypsinized primary endothelial cells. But the cells reattached to surface after several hours.
I did not see much differece between adherent and suspension transfection.


Hi, these are normal breast epithelial cells, but which are grown in suspension. Before transfection I trypsinize them and tried transfecting while they are still single cells. I havent yet tried by allowing them to attach as once I allowing them to attach will change the phenotype what I intend to study. Do you feel Lipofectamine could be better than Fugene? Is there any other factor which could be useful ?(Media is serum free)


lipofectamine holds a protocol for transfecting suspenson cell line. In combination with Plus Reagent, it gives pretty nice results. I attached both lipofectamine and plus reagent sheets.

here is the protocol for lipofectamine and suspension cell line
Transfecting Suspension Mammalian Cells
Use the following procedure to transfect mammalian cells in suspension in a
6-well format. All amounts and volumes are given on a per well basis.

1. On the day of transfection, prepare a single cell suspension from stock cells.
Wash the cells once with serum-free growth medium without antibiotics,
and seed cells at a density of 2-3 x 106 cells per well in 0.8 ml of serum-free
growth medium without antibiotics.
2. For each transfection sample, prepare complexes as follows:
a. Dilute 1-5 µg of DNA in 100 µl of Opti-MEM® I Reduced Serum Medium
(or other medium) without serum.
b. Mix Lipofectin® before use, then dilute 2-25 µl of Lipofectin® in 100 µl of
Opti-MEM® I Medium (or other medium) without serum. Let stand at
room temperature for 30-45 minutes.
c. Combine the diluted DNA with diluted Lipofectin® (total volume =
200 µl). Mix gently and incubate for 10-15 minutes at room temperature
(solution may appear cloudy).
3. Add the 200 µl of complexes to cells. Mix gently by rocking the plate back
and forth.
4. Incubate cells at 37°C in a CO2 incubator for 5-24 hours.
5. The following day, add 4 ml of complete growth medium to the cells.
6. Incubate cells at 37°C in a CO2 incubator for 24-48 hours prior to testing for
transgene expression.

you may also see these trheads ;

may give additional informations
selection of transfected suspension cells protocol
best time for G418 addition
antibiotocs during transfection


Hi Fred,
Thank you for the suggestion, links and the protocols, I will try to modify my protocol according to those and hope to see some green cells!