AMAXA PROTOCOL FOR TRANSFECTING HT-22 CELLS
CELL CULTURE BEFORE THE EXPERIMENT:
** Required medium: GIBCO, Cat. # 11995
+ 10% FBS
+ 1ml P/S (0.2%)
(1) Culturing of cells before nucleofection a.
Desired confluency of cells in a 75cm 2 flask should be 80%
PREPARATION OF SAMPLES:
** Make sure that you have made supplemented Nucelofector Solution V before continuing
Recipe:
Add 0.5 mL supplement to 2.25 mL Nucleofector Solution; mix
** Final sample should contain: gently. Stable for 3 months at 4 o C.
1 x 10
100 µ
6 cells
2 µ g plasmid DNA (or positive control pmaxGFP)
L Nucleofector Solution V
POSITIVE CONTROL:
Recommended amount of cells in Nucelofector solution but with pmaxGFP provided by the kit (2 µ g)
NEGATIVE CONTROLS:
Recommended amount of cells in Nucleofector solution but without application of program: i.
Cells + solution + DNA – program ii.
Cells + solution – DNA + program
(2) Prepare 2 µ g endotoxin free DNA for each sample
(3) Pre-warm supplemented nucelofactor solution V to room temp
(4) Pre-warm culture medium at 37 o C in a 50 mL tube
(5) Prepare 6-well plates by filling appropriate number of wells with 2 mL of culture medium a.
Pre-incubate plates in a humified 37 o C/5% CO
2
incubator.
(6) Remove media from HT-22 flask and add 5ml trypsin EDTA (Gibco Cat #
25200) of the HT-22 flask
(7) Incubate for ~ 3min or until cells float
(8) Add 5ml culture media to flask to inactivate the trypsin
(9) Place the media-cell suspension into a 50ml conical tube
(10) Spin down cells for 10min at 3000rpm
(11) Remove supernatant by sterile aspiration and resuspend pellet with 5ml fresh culture media by using a Pasteur pipette

(12) Add 400 µ l Trypan blue into a new 1.5ml eppendorf tube and add 100 µ l of the resuspended cell mixture a.
This is the dilution factor used for the calculation in step 14
(13) Shake eppendorf tube and place 21 µ l of the cell-trypan blue mix into an hemocytometer and count 4 quadrants
(14) Calculate the number of cells that is present in 1ml by performing the following calculation:
#cells x 5 (dilution factor) x 10000 = # cells in 5ml
# quadrants
#cells in 5ml = # cells in 1ml
5
Want 1000000 cells for transfection:
1000000 = ml of cell suspension needed to have 1000000 cells
# cells in 1ml
(15) Spin down cells (3000rpm, 10 min, RT)
(16) Remove supernatant by sterile aspiration
NUCLEOFECTION:
The next few steps have to take LESS than 15 min, so do them separately for each sample.
(17) Resuspend pellet with 100 µ l Nucleofector Solution V to a final concentration of
1x10 6 cells/100 µ l
(18) Add 2 µ l DNA into cuvette
(19) Be careful NOT to touch the metal parts of the cuvette
(20) Add 100 µ l of the cell-Nucleofector solution V into the cuvette and close cuvette with blue cap
(21) Give cuvette a flick of the wrist to mix DNA and cells and make sure that sample covers bottom of cuvette
(22) Make sure that there are NO bubbles in the cuvette
(23) Select Nucleofactor program U-030
(24) Place cuvette in cuvette holder and press the ‘X’ button to start
(25) Remove cuvette immediately upon completion of program (display shows
‘OK’)
(26) Transfer cells using plastic pipette in kit into a 15ml conical tube a.
This will prevent cell damage/loss
(27) Add 1ml HBSS into 15ml conical tube
(28) Rinse cuvette with 500 µ l HBSS and add it to the 15ml conical tube
(29) Allow cells to incubate for 10min at RT
(30) After 10min incubation time is over, add 500 µ l of the cell suspension into each well that contains 2ml of HT-22 media

a.
Want 50,000 cells per well
(31) Incubate cells in incubator over night
Determining Transfection efficiency
(32) Check for transfection efficiency the next day by using the fluorescent microscope
(33) Photodocument cells a.
Take Black and White pictures b.
Take pictures in which cells in the background and those that have been transfected are visible i.
Turn down light intensity, yet, do not turn light completely off c.
Take picture that show transfected cells only i.
Turn off entire background light
JS 10.29.07
JS 11.19.07