siRNA transfection in suspended cell lines MV4-11, THP-1 - (Jan/16/2007 )
Hi all... I'm presently trying to transfect siRNA into the Leukemia cell lines MV4-11, THP-1 with little success... Im using Lipofactamine 2000 transfection reagent and the siRNA's were purchased from Santa Cruz Biotechnoligies... The protocol doesn't seem to work well though... someone pls help me out... how do i optimise the conditions and how do i troubleshoot!
there is a protocol for lipofectamine and suspension cells.
please see it below :
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