HEK293T cells (American Type Culture Collection (ATCC), CRL-3216) and Neuro-2A cells (ATCC, CCL-131) were grown in DMEM plus GlutaMAX (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS at 37 °C with 5% CO2. Immortalized mouse astrocytes containing the APOE4 isoform of the human APOE gene (Taconic Biosciences) were grown in DMEM plus GlutaMAX (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS and 200 μg ml−1 Geneticin (Thermo Fisher Scientific). Cell lines were authenticated by their suppliers and were verified to be mycoplasma negative during the study.
Dmem plus glutamax
Manufactured by Thermo Fisher Scientific
Sourced in United States, Switzerland, United Kingdom
DMEM plus GlutaMAX is a basal medium for the cultivation of a wide variety of cells, including adherent and suspension cell lines. It contains L-glutamine, a critical component for cell growth and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (13 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Tryple express, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Hek293t, by American Type Culture Collection (3 mentions)
Dmem plus glutamax, by R&D Systems (2 mentions)
Poly d lysine, by Merck Group (2 mentions)
Trypsin edta 0.25, by Thermo Fisher Scientific (2 mentions)
Glutamax supplement, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
96 well flat bottom cell culture plate, by Corning (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Corning (2 mentions)
Glutamax, by Corning (2 mentions)
48 well poly d lysine coated plates, by Corning (2 mentions)
Le ma900 cell sorter, by Sony (2 mentions)
35 μm cell strainer, by Corning (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Hela tet off, by Takara Bio (1 mentions)
20 protocols using dmem plus glutamax
1
Cell Culture Conditions for HEK293T, Neuro-2A, and APOE4 Astrocytes
2
Cell Line Culture and Maintenance
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HEK293T (American Type Culture Collection (ATCC) CRL-3216), U2OS (ATTC HTB-96), K562 (CCL-243) and HeLa (CCL-2) cells were purchased from ATCC and cultured and passaged in DMEM plus GlutaMAX (Thermo Fisher Scientific), McCoy’s 5A medium (Gibco), RPMI medium 1640 plus GlutaMAX (Gibco) or DMEM plus GlutaMAX (Thermo Fisher Scientific), respectively, each supplemented with 10% (v/v) FBS (Gibco, qualified). Cells were incubated, maintained and cultured at 37 °C with 5% CO2. Cell lines were authenticated by their respective suppliers and tested negative for mycoplasma.
3
Immortalized Mouse Embryonic Fibroblasts for Genetic Analyses
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Primary MEFs were generated from E14.5 embryos by standard methods. The LARP4 KO MEFs were described (Mattijssen et al., 2017 (link)). Each MEF cell line was derived from a different embryo, all females. The MEFs used to generate the data in Figure 3 were immortalized as described using SV40 Large-T antigen (Mattijssen et al., 2017 (link)), the experiment in Figure 2 was performed using three independent WT- and 3 LARP4 KO MEF cell lines (N = 3, biological replicates). MEFs used for the IFNα ActD experiment in Figure 5 were cultured following the 3T3 protocol for spontaneous immortalization (Todaro and Green, 1963 (link)). The experiment in Figure 5 was performed using two independent WT- and 2 LARP4 KO MEF cell lines (N = 2, biological replicates). The immortalized MEFs and HEK293 cells were cultured in DMEM plus Glutamax (Gibco) supplemented with 10% heat-inactivated FBS (Atlanta Biologicals). HEK293 cells are not commonly misidentified. Nonetheless DNA from these cells was authenticated by ATCC via STR (short tandem repeat) profiling. Standardized testing (ATCC) had verified that the cells were free of mycoplasma infection.
4
HeLa and HEK293 Cell Culture
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HeLa Tet-Off (Clontech) and HEK293 were maintained in DMEM plus Glutamax (Gibco) supplemented with 10% heat-inactivated FBS (Atlanta Biologicals) in a humidified 37°C, 5% CO2 incubator. Cultures were passed every 2–3 days. The HeLa Tet-Off cells were a gift obtained directly from Gerald Wilson (U Maryland, Baltimore). HEK293 cells were obtained from Tazuko Hirai in Bruce Howard's laboratory at the NIH. Our HEK293 and HeLa cell DNAs were both authenticated by the ATCC via STR profiling. HeLa Tet-Off cells are not commonly misidentified cell lines as listed by the International Cell Line Authentication Committee. Standardized mycoplasma testing (ATCC) was performed and tested negative.
5
Isolation and Culture of Primary Schwann Cells
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Primary Schwann cells cultures were obtained from sciatic nerves harvested from adult Sprague Dawley rats. The nerves were collected in cold DMEM plus glutamax (Gibco, Carlsbad, CA, USA) containing 100 U/mL penicillin (R&D system, Minneapolis, MN, USA) and 100 g/mL Streptomycin (R&D system, Minneapolis, MN, USA). Nerves were then gently dissected and the epineurium removed with fine forceps. Fragments of nerves were then plated in 35 mm petri dishes and incubated with glial permissive medium (DMEM plus glutamax, 100 U/mL penicillin and 100 g/mL Streptomycin, 14M forskolin and 100 ng/mL LNRG11; R&D System, Minneapolis, MN, USA) for 2 weeks under culture conditions (37°C, 5% CO2). The medium was replenished every 72 hours. The culture medium was then enriched with 0.125% collagenase type IV (ThermoFisher, Lafayette, CO, USA) and 117 U/mg dispase (Gibco) for 24 hours and mechanically dissociated with a Pasteur glass pipette. The cell suspension was filtered through a 70 µm cell strainer (BD Biosciences) and centrifuged at 100 × g for 5 minutes. The cell pellet was resuspended in glial permissive medium, seeded on 35 mm petri dishes pre-coated with poly-D-lysine (Sigma) and incubated under culture conditions. An antibody complement method was adopted to purify Schwann cells from fibroblasts (Tohill et al., 2004; Kaewkhaw et al., 2012; Pascal et al., 2014).
6
Generating Immortalized Mouse Embryonic Fibroblasts
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MEFs were generated from E14.5 embryos from the same litter by standard methods. Each MEF cell line was derived from a different embryo. MEFs were derived from KO and WT matched siblings, all females. MEFs at passage 3 were transfected with SV40 Large-T antigen-expressing plasmid pBSSVD2005 (Addgene plasmid 21826) using Lipofectamine 2000 (Invitrogen) and subcultured at 1:10 for at least 5 passages. Cells were maintained in DMEM plus Glutamax (Gibco) supplemented with 10% heat-inactivated FBS (Atlanta Biologicals) in a humidified 37°C, 5% CO2 incubator. Cultures were passed every 2–3 days.
7
HEK293 Cell Transfection Protocol
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HEK293 cells were maintained in DMEM plus Glutamax (Gibco) supplemented with 10% heat-inactivated FBS in humidified 37°C, 5% CO2 incubator. The cells were transfected using Lipofectamine 2000 according to manufacturer’s instructions using 2.5 ug plasmid DNA and 6.25 ul lipofectamine on cells that were 80% confluent in 6-well plates. The next day, cells were seeded onto coverslips for confocal microscopy (see below), or divided over multiple 10 cm plates for mitochondrial preparation (see below) and western blot analysis.
8
Ex Vivo Model of Dermal-Epidermal Separation
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DES was analysed using an ex vivo model of DES as previously described (19) with minor modifications. Briefly, human foreskin was washed and embedded in an optimum cuttingtemperature compound (Tissue-Tek® O.C.T. ™ compound, Sakura Finetek Europe B.V, Alphen aan den Rijn, The Netherlands) and stored at -20°C. Six 6-µm thick cryosections were placed on adhesive microscope slides (Starfrost®, Medite Service AG, Dietikon, Switzerland). BPS and NHS were diluted 1:2 with PBS and applied to the skin sections for 2 h at 37°C, followed by washing the slides with PBS. Eosinophils were resuspended in DMEM (DMEM plus GlutaMAX™, Gibco®, Life Technologies Europe B.V., Zug, Switzerland), including 10% foetal calf serum to avoid their immediate adherence to the slides, and subsequently added to the sections in the prepared chambers for an incubation of 4 h at 37°C. Tissue sections were then fixed with 3.7% formalin and stained with hematoxylin and eosin (H&E). DES was evaluated by light microscopy studies. The total lengths of DES and DEJ, respectively, were calculated using Fiji (23) . DES was given as a percentage of separation at the DEJ. To assess tissue damage semi-quantitatively in indicated experiments, we scored the extent of skin damage and presence of DES. Each experiment was repeated at least three times to confirm reproducibility.
9
Optimizing Cell Culture Conditions
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Penicillin‐streptomycin, L‐glutamine, DMEM plus GlutaMAX, and minimum essential medium (MEM) non‐essential amino acids were from Invitrogen (Stockholm, Sweden), whereas Dulbecco Modified Essential Medium (DMEM), all salts for buffers, and the anti‐DNP IgE antibody were from Sigma (Stockholm, Sweden). DNP‐HSA was from Biosearch Technologies (Petaluma, CA). BET protein inhibitors PFI‐1 (PF‐6405761), I‐BET151 (GSK1210151A), and I‐BET762 (GSK525762) were from Sigma.
10
Schwann Cell Isolation from Rat Sciatic Nerve
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To harvest Schwann cells (SC), rat sciatic nerves were exposed, removed, and kept in DMEM plus glutamax (Invitrogen, UK) containing 100 U/mL penicillin and 100 μg/mL streptomycin. Nerves were then dissected in trunks, desheathed, and finally chopped in 1 mm segments. The segments were then plated in a Petri dish in SC growth medium (DMEM plus glutamax containing 100 U/mL penicillin, 100 μg/mL streptomycin, 14 μM forskolin, and 100 ng/mL NRG1β1, R&D Systems, UK). Cells were incubated for 2 weeks at 37°C with fresh medium added approximately every 72 h. After these 2 weeks medium was aspirated and 0.125% (w/v) collagenase type IV and 117 U/mg dispase were added to the Petri dish. After 24 hours (h) incubation, cell suspension was filtered through a 70 mm cell strainer (Falcon; BD Biosciences Discovery Labware, Bedford, MA) and centrifuged at 100 ×g for 5 min to obtain the cell pellet. Finally, the cell pellet was resuspended in SC growth medium, seeded into a Petri dish pre-coated with poly-D-lysine (Sigma, St Louis, MO, USA), and incubated in the same conditions. The following day, the medium was changed and cells were left to proliferate. When confluent, SC were purified by an antibody complement method to eradicate the remaining fibroblasts [14 (link)–16 (link)].
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