Human lung adenocarcinoma A549 cell line was obtained from American Type Culture Collection (ATCC, CCL-185™). Human colorectal adenocarcinoma DLD-1 (ATCC, CCL-221™) cell line was provided by Dr L Grumolato (DC2N laboratory, Inserm U1239, Mont-Saint-Aignan, France) and human embryonic kidney HEK-293 (ATCC, CRL1573™) cell line was generously given by Dr Prézeau (IGF laboratory, Montpellier, France). All cell lines were routinely maintained according to the instructions from ATCC. More precisely, A549 and DLD-1 cells were cultured with RPMI 1640 media and HEK-293 cells were cultured with DMEM media, all supplemented with 1% sodium pyruvate (ThermoFisher Scientific, Montigny-Le-Bretonneux, France) and 10% foetal bovine serum (FBS, Lonza, Levallois-Perret, France). Transient transfections were performed using either Amaxa® Cell Line Nucleofactor® Kit V (Lonza, Levallois-Perret, France) or FuGene® HD (Promega Corporation, Southampton, UK) according to the manufacturer’s protocol.
Amaxa cell line nucleofactor kit 5
Manufactured by Lonza
Sourced in France, Switzerland
The Amaxa cell line nucleofactor kit V is a laboratory equipment product designed for the transfection of cells. It facilitates the introduction of nucleic acids, such as plasmids or small interfering RNAs, into various cell lines. The product enables efficient gene delivery and gene expression studies.
Automatically generated - may contain errors
Lab products found in correlation
Pgl4.23 luc2 minp vector, by Promega (2 mentions)
Dual glo luciferase, by Promega (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Ccl 221, by American Type Culture Collection (1 mentions)
Crl 1573, by American Type Culture Collection (1 mentions)
Fugene hd, by Promega (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Ccl 185, by American Type Culture Collection (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
4d nucleofector core unit, by Lonza (1 mentions)
Facsaria fusion, by BD (1 mentions)
Nucleofector 2b device, by Lonza (1 mentions)
2 deoxyglucose, by Merck Group (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
7 protocols using amaxa cell line nucleofactor kit 5
1
Cell Culture and Transfection Protocols
2
Hypoxic and Normoxic Cell Fluorescence Tagging
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To analyze the interaction between hypoxic and normoxic cultured cells, we stably inserted the coding sequence for the green or red fluorescent protein (GFP or RFP) into C2C12 myoblasts. Therefore, 0.4 µL of sleeping beauty transposon plasmids (GFP: pSBbiGP; RFP: psBbiRP) [13 (link)], 0.9 µg transposase (pCMV(CAT)T7-SB100) [14 (link)], and 20 µL nucleofector solution (Amaxa™ Cell Line Nucleofactor™ Kit V, Lonza, Basel, Switzerland) were added to 500,000 cells and electroporated with the 4D-Nucleofector™ Core Unit (program B 032, Lonza, Basel, Switzerland). After nucleofection, cells were seeded in 6-well plates, selected with 2 µg/mL puromycin (Thermo Fisher, Waltham, MA, USA) for 10 days under normoxic condition and then sorted for high fluorescence (FACSAriaFusion, BD, Franklin Lakes, NJ, USA). For all further experiments, C2C12GFP were exclusively cultured at 21% O2, while C2C12RFP were cultured at 2% O2.
3
WASP Gene Knockout via CRISPR/Cas9 Transfection
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For transfection, cells were cultured without antibiotic 1 day before transfection. 3 × 106 cells were harvested at 90 × g for 10 min and resuspended with 100 µl of Amaxa cell line Nucleofactor Kit V (VCA-1003) or L solution (VCA-1005) (Lonza, Basel, Switzerland). 2 µg of plasmid (WASP-CRISPR/CAS9-GFP for WAS gene knock out, pCMV6-AC-mGFP-WASp-WT or pCMV6-AC-mGFP-WASp-∆RBM1 for transfection) was added to cell suspension, incubated at room temperature for 10 min, transferred to cuvette and transfection performed using nucleofector 2b device (Lonza, Basel, Switzerland). Transfected cells were cultured for 1 day and then subjected to FACS for sorting GFP-enriched cell population.
4
Translocated Enhancer Sequences and Controls
5
Imaging H2B-GFP and H2B-mCherry in MDA MB 231 Cells
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H2B–GFP (from Addgene vector 11680 ) and H2B–mCherry (from Addgene vector 20972 ), were introduced into MDA MB 231 cells by electroporation using an Amaxa Cell Line Nucleofactor® Kit V (Lonza, VCA-1003). 2 µg of DNA was introduced into cells according to manufacturer's instructions. Transfected cells were seeded on 35 mm glass-bottom dishes and left for 72 h in standard tissue culture conditions. After incubation, cells were imaged using FLIM microscopy. For 2-deoxyglucose (2-DG) and sodium azide treatment, cells were treated with 50 mM 2-deoxyglucose (Sigma, D8375) and 10 mM sodium azide (Sigma, S8032) 30 min before acquiring images. Control cells were treated with H2O.
FLIM images were taken on a Nikon TE 2000 inverted microscope fitted with a Lambert Instruments FLIM Attachment (LIFA) and a Yokogawa CSU22 spinning disk unit with 100× objective. The microscope was equipped with an incubation chamber suitable to maintain live cells and optics at constant temperature. The LIFA system was equipped with an Omicron 50 mW 445 nm laser for CFP lifetimes and a 60 mW 488 nm laser for GFP lifetimes. The experiment is based on the frequency domain method for fluorescence lifetime imaging and allows the rapid acquisition and generation of lifetime images. Fluorescence lifetimes were measured using LI-FLIM software (Lambert Instruments).
FLIM images were taken on a Nikon TE 2000 inverted microscope fitted with a Lambert Instruments FLIM Attachment (LIFA) and a Yokogawa CSU22 spinning disk unit with 100× objective. The microscope was equipped with an incubation chamber suitable to maintain live cells and optics at constant temperature. The LIFA system was equipped with an Omicron 50 mW 445 nm laser for CFP lifetimes and a 60 mW 488 nm laser for GFP lifetimes. The experiment is based on the frequency domain method for fluorescence lifetime imaging and allows the rapid acquisition and generation of lifetime images. Fluorescence lifetimes were measured using LI-FLIM software (Lambert Instruments).
6
Transient Transfection of EL4 Cells
7
Translocated Enhancer Sequences and Controls
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Five translocated enhancer sequences and five controls with scrambled MYB consensus motifs (replacing CNGTT with GTAAG, see Supplementary Table 6 ) were synthesized and cloned into the pGL 4.23 [luc2/minP] vector (Promega) by BlueHeron. Enhancer activity was measured in 6 replicates as relative luminescence of the pGL 4.23 [luc2/minP] vector compared to the pGL 4.73 [hRluc/SV40] with Dual-Glo Luciferase (Promega) after a 36 hour co-nucleofection into Jurkat cells following the manufacturer’s instructions (Amaxa cell line nucleofactor kit V from Lonza).
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