How to handle and transfect suspension cells? - (Feb/09/2005 )

Hi,
I am going to handle some suspesion cell lines and do transfection. I never play with suspension cells. can anyone tell me how to culture it and how to do transfection in suspenstion cells???

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I think electroporation is the best way to transfect suspension cell.

Simon

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Hi
there was a discussion about it at
- Forum: siRNA and RNAi · Post Preview: #10641

For my experiment, i foud a protocol on the user maual of transfection reagent. here is the protocol for lipofectamine :

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.

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Thank you all for your reply.
As I never handle suspension cell, so I don't know some basic things, such as how to culture , how to distinguish the dead cells from living cells, how to seperate them when passaging cell ? Is there any tip during culture??


Thanks !!!!

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I've been using THP-1 cells, which are a suspension cell. If you're just passing them, you can take out some of the media with cells from one plate and add it to fresh media in another plate. If you're going to plate them for an experiment you'll want to spin them down gently and resuspend in a serum free (or starvation) media at the cell density you want. As far as live/dead counts, our lab usually uses Trypan Blue exclusion, so blue cells are dead and the others are living. For THP-1 cells, the lab usually will pass them at a 1:2 dilution if the number of cells per ml is between 5 and 8 million cells. If there's more than 8 million cells per ml, then they're usually passed at a 1:3 dilution. Hope this helps at least a little bit.

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Hi Fred, I was wondering if the 6-well plate you were talking about is a low attachment plate! For cells in suspension how you would prevent them to attach during those 24-48 hrs? (Even in serum free media wont the cells attach?) Thanks

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We grow our suspension cells in Techne stirrer bottles which over the past 20 years have proven to be successful. For cells like J774 and RAWs which can be grown as adherent cells as well as suspension cells the advantages of stirrer bottles are as follows :-

a) The cells are kept in suspension by stirring which also reduces aggregation.

You can grow vast numbers in a very small footprint i.e. a 500ml stirrer can yield 400 million cells which is roughly equivalent to 20 x T175cm flasks.

c) To subculture all you need to do is pour off the cells you need for the experiment and replace with fresh media.

d) You save money on consumables such as TC flasks ( as the stirrer bottles are glass and reusable), PBS and Trypsin + media are not required for subculture.

e) The cells because they have not been trypsinised still express membrane receptors and the numbers of viable cells are always high i.e. 99% on average.


I only do very few transfections and these are always on adherent cells, so sorry no advice on that.

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the protocol is ok for true suspension cells. for cell that attach low, i usethe standard protocol that works better.
But in suspension cells, we have a stiring bottle

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