Biology Reference
In-Depth Information
4.
Add the DNA/CaCl 2 mixture to 0.5 mL 2XHBS dropwise with vigorous bub-
bling using an automatic pipetter (keep eject button depressed) for 15-45 s (the
length of bubbling time depends on each batch of 2XHBS).
5.
Add HBS/DNA solution dropwise onto media, gently and quickly, by spreading
across cells in media.
6.
Observe the cells under a microscope. Good transfection efficiency is expected
when the particle size is small and uniform. Big or aggregated particle usually
indicates that the pH is not optimized, resulting in a poor transfection efficiency.
7.
C incubator and shake plates back and forth to distribute DNA/
CaPhosphate particles evenly.
Put plates in 37
°
8.
After 24 h, change the media to 5 mL fresh DMEM containing 10% FBS (virus is
more stable if incubation is carried out at 32
°
C).
3.4. Infection of Target Cells
1.
Split the appropriate cells (which you would like to infect) at 200,000 cells per 60 mm
plate in 2 mL of appropriate culture media. For suspension cells, they should be
growing in a log phase at the time of infection (for Jurkats, ideal density at time
of infection is 5
×
10 5 /mL).
2.
NX cells 24-48 h after transfection. Cen-
trifuge the supernatant at 1000 g for 5 min to pellet cell debris and filter through
0.45 Millipore filter to remove cells as well. Add Polybrene (4
Collect supernatant from transfected
Φ
µ
g/mL). Virus
containing supernatant can be frozen at -80
°
C for later infection ( see Notes 5 ).
3.
Remove 1 mL of media from each plate with cells you are going to infect.
4.
Add 4
g/mL polybrene (stock solution is 4 mg/mL) to each plate them with
gentle shaking.
µ
5.
Add 1 mL viral supernatant to cells plated for infection and place them at 37
°
C
with gentle shaking. For suspension cells, pellet 5
10 5 suspension cells and
resuspend cell pellets in 1 mL of viral supernatant containing 4 mg/mL polybrene.
Spin cells and wash away virus supernatant after incubation for 8-24 h. Suspen-
sion cells, especially some B-cells and T cells, are sensitive to polybrene and it
may be necessary to titrate polybrene.
×
6.
Remove virus 24 h after infection by feeding cells with fresh medium. For sus-
pension cells, spin down cells and resuspend in 2 mL of fresh media.
7.
After 24-48 h of infection cells are ready to assay for biochemical event of inter-
est. The reverse transcription and integration take place within 36 h, depending
on cell growth. Expression can be observed at 24 h, usually reaching its maxi-
mum at 48 h. For the Tc-regulatable retroviral system, add or remove tetracycline
or doxycycline depending on Tet-on or Tet-off viral vector, respectively.
3.5. Determination of the Viral Titer
The viral titer produced by packaging cells is determined as follows.
1.
Prepare the target cells by plating one day before infection into a six-well plates,
5
10 4 - 1
10 5 cells per well)
×
×
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