monocyte transfection

StewJW stewjw at gmail.com
Wed Aug 30 03:19:51 EST 2006


This protocol is taken from Invitrogen's web site for transfection of
THP-1 human monocytes.  Its uses:

The new transfection reagent Lipofectamine LTX (15338-100, 1ml)
Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062)
PLUS™ Reagent (Cat. No. 11514-015)


Use this procedure to transfect plasmid DNA into THP-1 cells in a
24-well format (for other formats, see Scaling
Up or Down Transfections, below). All amounts and volumes are given on
a per well basis.
1. The day of transfection, count the cells to determine culture
density. Plate 1 x 105 cells per well in 0.5 ml of
complete growth medium. Cell density should be 50~80% confluent on the
day of transfection.
2. For each well of cells to be transfected, dilute 0.5 µg of DNA into
100 µl of Opti-MEM® I Reduced Serum
Medium without serum.
3. If using PLUS™ Reagent: Mix PLUS™ Reagent gently before use,
then add 0.5 µl PLUS™ Reagent (a 1:1 ratio to
DNA) directly to the diluted DNA. Mix gently and incubate for 5-15
minutes at room temperature.
4. For each well of cells, dilute 1.25-2.25 µl of Lipofectamine™ LTX
into the above diluted DNA solution, mix
gently and incubate for 25 minutes at room temperature to form
DNA-Lipofectamine™ LTX complexes.
5. Add 100 µl of the DNA-Lipofectamine™ LTX complexes directly to
each well containing cells and mix gently
by rocking the plate back and forth.
6. Complexes do not have to be removed following transfection. Incubate
the cells at 37°C in a CO2 incubator
for 18-24 hours post-transfection before assaying for transgene
expression.

I work for Fisher UK which supplies these reagents.



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