HEK293 transfection – MATra
Various methods how deliver exogenous DNA into mammalian cells have been developed. The protocol below describes transfection procedure of HEK293 cells based on the MATra (Magnet Assisted Transfection). Using this technique exogenous DNA is in the first step associate with magnetic nanoparticles. Subsequently established complexes are delivered into the individual cells applying strong magnetic field.
Chemicals:
OptiMEM – Invitrogen 11058-021
Fetal bovine serum (FBS) – Invitrogen 10108-165
MATra Reagent – IBA 7-2001-100
Collagen – Serva 47254
P-L-Lysine – Sigma P9155
Trypsin –Sigma T4799
DL-APV – Sigma A5282
Kynurenic acid (KYN) – Sigma K3375
Ketamine – Vetoquinol “Narketan 10”
Solutions:
Collagen 0.5 mg/ml – one vial (20 ml) diluted with 60 ml of water
PLL 10 ug/ml - one vial (5 mg) dissolved in 500 ml of 0.15 M boric acid
HEK293 culture medium – OptiMEM supplemented with FBS (5 % vol.)
PBS – 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, adjust pH to 7.3
Trypsin 0.05 % – 25 mg trypsin, 10 mg EDTA, in 50 ml of PBS
APV – 59 mg APV, 170 mg KYN, 1 ml 1 M NaOH, water to final vol. 30 ml, adjust pH to 7.3
Ketamine 50 mg/ml – 10 ml of Narketan diluted with 10 ml of water
MgCl2 1 M – 20.3 g MgCl2.6H2O dissolved in 100 ml of water
Protocol:
a/ Collagen+PLL coverslips
Clean glass coverslip in 1 M HCL, rinse them extensively with water and finally wash in 70 % ethanol. Allow coverslips to dry and sterilize them by autoclaving.
Individual coverslips coat firstly by small amount of collagen and allow coverslips to dry overnight. Next day cover surface of coverslips with sufficient amount of PLL solution and incubate them at least for 1 hour. Then rinse coverslips 3 times with water and store them in room temperature up to two weeks.
b/ HEK 293 cultures
HEK293 cells are cultured in OptiMEM medium supplemented by 5% FBS without antibiotics. One day before transfection harvest confluent parental cell culture and split cells. We seed 200,000 cells per one well in 24-well plate with PLL coated surface.
c/ Transfection of NMDA receptors
Plasmid DNA, carrying gene of interest, intended for transfection should be izolated as an endotoxin free (e.g. EndoFree kits, Qiagen). We do co-transfection genes of un-tagged NMDA receptor subunits and “empty” EGFP expressing vector to identify successfully transfected cells.
Firstly for each transfection mix add 50 ul of OptiMEM (without serum) into eppendorf tube, then add DNA (250 ng of each NMDA subunit – NR1 and NR2, 200 ng of EGFP) and finally 0.9 ul MATra reagent (vortex well before use). Flick several times to mix and briefly spin down. Leave at room temperature for 20 min to establish transfection complexes. Meantime for each transfection prepare one or two 3.5 cm Petri dishes containing 1 ml OptiMEM, 100 ul APV, 15 ul MgCl2, 15 ul FBS and 4 ul ketamine. Equilibrate medium in incubator. In addition, pre-warm PBS and trypsin solutions in water bath set at 37 °C.
After 20 min incubation of DNA with MATra reagent add each mixture dropwise to the HEK293 cells cultured in 24-well plate. Transfer plate on the Magnet Platform and incubate for 15 min.
Once magnet-incubation is finished remove all medium from cells, wash them gently with PBS and add 150 ul of trypsin solution. Monitor cell detachment under microscope. Triturate cells quickly and transfer them in prepared Petri dishes. Maintain cultures overnight in an incubator, next day check the transfection rate (EGFP expression) and proceed with an electrophysiological experiment.