Page 56 - GPD-2-2

Gene & Protein in Disease Transfection methods for TK6 cells

seeded in a 6-well plate at a density of 1 × 10 cells/mL. 2.6. Cell nucleofection
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After that, the cells were transfected with 0.5 µg and 1.0 µg 2.6.1. Initial nucleofection optimization
pGFPmax using Metafectene Pro (Biontex, Germany) at a
ratio from 1:2 to 1:6; control cells were not transfected with Nucleofection was carried out using the Jurkat Cell
DNA. Transfection was accomplished by adding the DNA Line Optimization 96-well Nucleofector Kit from
and Metafectene to solutions A and B, respectively, with Amaxa (catalogue no: V4XC-1024), according to the
both containing minimal essential medium (MEM) with manufacturer’s recommendations. Briefly, TK6 cells were
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a reduced serum 5% (OptiMEM) instead of the standard split into three aliquots, each containing 1.0 × 10 cells. The
10% FBS. Solutions A (DNA + OptiMEM + Glutamax) aliquots were centrifuged at 0.2 g RCF for 5 min at room
and B (Metafectene Pro + OptiMEM + Glutamax) were temperature, and the media was completely removed.
mixed and incubated at room temperature for 20 min. Each of the three cell pellets was resuspended in one of
After incubation, the DNA-lipid complexes were added three different nucleofection solutions (SE, SF, and SG),
dropwise to the cells and swirled with extreme care to and 0.4 µg pmaxGFP plasmid (Lonza) that encodes green
avoid breaking up the complexes. The samples were kept fluorescent was added to each solution. Each well in the
in a CO incubator at 37°C. GFP and cell viability were 96-well nucleofection plate contained 20 µL of cells and
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assessed at 24 and 36 h post-transfection. DNA in one of the three nucleofection solutions; control
cells received no DNA. Immediately, the mixture was
In a parallel experiment, the cells were transfected
with pNl1.1 CMV expressing NanoLuciferase, and control transferred into an Amaxa Shuttle nucleofection conducted
using the recommended program. On completion of the
cells were transfected with the empty pNL1.2 vector. Cells nucleofection program, 80 µL of pre-warmed complete
transfected with no DNA served as an additional control. media was added to each well of the 96-well Nucleocuvette
The ratio of DNA (in µg) to lipid-mediated reagent varied plate. The contents (100 µL) of each Nucleocuvette well
from 1:2 to 1:6. Solutions A (DNA + OptiMEM + Glutamax)
and B (Metafectene Pro + OptiMEM + Glutamax) were were rapidly removed and transferred to the appropriate
cell culture plates. The cells were incubated for 24 – 36 h
mixed and incubated at room temperature for 20 min. The in a humidified 37°C/5% CO atmosphere. Transfection
DNA-lipid complexes were added dropwise to the cells and efficiency was determined by fluorescence microscopy,
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swirled with extreme care. The samples were kept in a CO and cell viability was assessed using the Vi-CELL counter
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incubator at 37°C. NanoLuciferase Assay (Promega) was (Beckman). Unless otherwise indicated, all nucleofection
performed at 24 and 36 h post-transfection by following experiments were carried out in duplicate and repeated
the manufacturer’s protocol. Transfection efficiency using twice.
normalized luciferase activity and GFP, as well as cell
viability, was determined at 24 and 36 h post-transfection. 2.6.2. Secondary nucleofection optimization
The experiment was conducted in duplicate and repeated
twice. A second nucleofection optimization was performed
using SF reagent, which allowed the further evaluation
2.5. Lipofectamine transfection of this reagent. TK6 lymphoblastic cells were pelleted
by centrifugation at 0.2 g RCF for 5 min at room
Transfection was performed according to the temperature. The cells were resuspended to a density of
manufacturer’s protocol by following the optimized 1.0 × 10 cells/20 µL in the SF-supplemented nucleofection
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protocol for Jurkat cells. TK6 cells were seeded in 6-well solution (Lonza). The p53-proficient TK6 cells were
plates at a density of 1.0 × 10 and 1.0 × 10 cells/well. nucleofected with 0.4 µg pGFPmax (0.5 µg pGFPmax was
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Different concentrations of pGFPmax/empty pNL1.2 (0.5
– 1.0 µg) were diluted into 100 µL optiMEM media, PLUS also tested); control cells were not transfected with DNA.
reagent (1.5 – 2.5 µL), and lipofectamine (3.75 – 10.00 µL). In a parallel experiment, the cells were transfected
After 30 min of incubation at room temperature, 100 µL of with 0.4 µg and 0.5 µg pNL1.1. CMV and control cells
the DNA/PLUS/Lipofectamine LTX complexes were added were transfected with the promoter-less vector pNL1.2;
to the cells in complete growth media in the 6-well plate additional controls were cells that did not receive DNA.
and incubated at 37°C in the 5% CO /95% air incubator. Nucleofection was conducted using the DS 137 program
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Control cells received no DNA. Cells transfected with on the Amaxa Nucleofector 96-well Shuttle. After
an empty pNL1.2 vector served as an additional control. nucleofection, the contents of each microcuvette (Lonza)
GFP transfection efficiency, normalized NanoLuciferase well were rapidly removed with 80 µL of pre-warmed
activity, and cell viability were determined at 24 and 36 h RPMI 1640 media supplemented with 2 mM L-glutamine
post-transfection. The experiment was conducted in and 10% FBS and transferred to the appropriate wells in the
duplicate and repeated twice. 6-well culture plates. The cells were incubated for 24 – 36 h

Volume 2 Issue 2 (2023) 3 https://doi.org/10.36922/gpd.0353