Prepared by:
Elizabeth D. Chao
Kryn Stankunas
Crabtree Laboratory
Date: August 30, 1999

JURKAT TRANSFECTION PROTOCOL USING THE ELECTROPORATION METHOD

Background:

Transfection, the introduction or delivery of nucleic acids into cultured mammalian
cells, has provided a powerful way to analyze the function, regulation, and interaction of
mammalian genes along with their gene products. Many methods have been developed to
facilitate the chemical and physical transfection of nucleic acids into mammalian cells,
including DEAE-dextran, calcium phosphate, liposome-mediated transfection, micro-
injection, and electroporation. The technique that we describe here utilizes electroporation,
a method which exposes the cells to a brief, defined electrical pulse to create transient pores
to allow plasmid DNA to cross the cell membrane.

Cell line used for this procedure:
(Leukemia cell line)

TAg (SV40 large T antigen) Jurkat T lymphocytes

Materials:

TAg Jurkat cell media:

Preparing the Media:
(Prepared under a sterile hood)

Fresh RPMI 1640 (Bellco Biotech, pH 7.3) supplemented with:
1%1 M HEPES pH 7.4
2 mML-Glutamine
100 units/ml Penicillin G
100 ug/mlStreptomycin
10% (v/v)Fetal Bovine Serum (GIBCO BRL)
Mix well + filter
Pre-warm to 37ºC prior to Jurkat transfection

IMAGE index01.gif

6-well flat-bottom multiwell Falcon plates
96-well flat-bottom microtiter plates
250 ml sterile polypropylene Corning centrifuge tubes with plug seal caps
UV Sterilized electroporation cuvettes (gap width 0.4 cm)

DNA Concentration for Transfection: Plasmid DNA used for transfection should be
highly purified, sterile, and free from contaminants such as endotoxins. We suggest that the
plasmid DNA should be prepared by 2X CsCl or Qiagen. Although the optimal DNA
concentration for each transfection may vary, we recommend using 2 µg of purified DNA as
a starting point.

Pre-aliquot DNA for transfection:
Pre-aliquot the appropriate volumes of target plasmid and reporter into sterile microfuge
tubes. This facilitates the later step of adding the DNA directly to the cell culture, especially
when many transfections are being performed simultaneously.

Cell Culture Density at time of transfection:
Approximately 107cells should be used per Jurkat transfection. A good density for
transfection is usually 5.5 x 105cells/ml. Therefore, for a cell density of 5.5 x 105 cells/ml:

Use:

(107÷ 5.5 x 105) x (# of transfections)

mls of Jurkat cell culture

Pre-transfection:

Spin the proper volume of cell culture down in a 250 ml Corning centrifuge tube
(plug-seal cap) at 1K for 5 minutes to pellet the cells.

While waiting for cells to spin down, aliquot 10 ml of pre-warmed Jurkat cell
culture media to each well in the 6-well flat-bottom multiwell Falcon plates

Aspirate the media from the pelleted cells

Use 300 µl of media per transfection. Depending on the number of transfections,
use:
300 µlx (# transfections) mls to resuspend the pellet

IMAGE index02.gif

Add 300 ul of resuspended cells to pre-aliquoted target plasmid + reporter

Pipet to mix well

Transfer to electroporation cuvettes

Electroporation (using the Bio-Rad Gene Pulser)

Electroporation volume: 300 µl
Set the appropriate voltage (for Jurkat cells, 250 Volts)
Check the capacitance (960 µF)
Gap Width: 0.4 cm
To zap, press both buttons down and wait
Flick the cuvettes after zapping to mix contents

Post-transfection:

Transfer the electroporated Jurkat cells to 6-well flat-bottom multiwell Falcon plates
containing the pre-aliquoted media.

Incubate transfected cells in the incubator (5% CO2at 37ºC and 95 % humidified
atmosphere)

Harvest cells after 24 h
Perform the desired assay (Secreted Alkaline Phosphatase Assay used in the following
protocol)
References:
1)Berger, J., Hauber, J., Hauber R., Geiger, R., and Cullen, B.R. (1988) Gene (Amst) 66, 1-
10
2)Cullen, B.R., and Malim, M.H. (1992) Methods Enzymol. 216, 362-368.

Aliquot harvested cells in triplicate into 96-well flat-bottom microtiter plates (2 x 105
cells/well in 100 µl of complete medium)

Stimulate with ionomycin (1 µM) and phorbol myristate acetate (PMA, 20 ng/ml) in a
final volume of 200 µl

Reporter gene activity was measured 12-24 h after stimulation.

Secreted alkaline phosphatase activity was measured 16-24 h after stimulation

Microtiter plates were heated to 65ºC for 1.5-2.0 h

100 µl aliquots from each well were incubated with an equal volume of 2 M
diethanolamine bicarbonate (pH 10.0), 1 mM methylumbelliferyl phosphate (Sigma) at
37ºC.

Relative alkaline phosphatase activity was measured by quantitating the accumulation of
flourescent product using a Titertek Fluoroskan II (ICN) with an excitation wavelength
of 355 nm and an emission wavelength of 460 nm

Preparation of cell extracts:

References:
Fiering, S., Northrop, J.P., Nolan, G.P., Mattila, P.S., Crabtree, G.R., and Herzenberg, L.A.
(1990) Genes & Dev. 4, 1823-1834

Cells were washed once with cold phosphate buffered saline
Resuspended in buffer A (10 mM Hepes (pH 7.8)), 15 mM KCl, 2 mM MgCl2, 1 mM
dithiothreitol, 0.1 mM EDTA
Pelleted by low speed centrifugation (Eppendorf microcentrifuge, setting 3 for 3 min)
Re