How to handle suspension cells?
Posted 09 February 2005 - 04:58 PM
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???
Posted 10 February 2005 - 07:50 AM
Posted 10 February 2005 - 08:26 AM
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
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
Posted 10 February 2005 - 06:15 PM
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??
Posted 28 February 2005 - 08:57 AM