All the IDH1 mutant pUltra-Chili transfer plasmids were packaged into lentiviral vectors (LVs) by transient transfection of HEK293T cells along with the 3 packaging plasmids: pVSVg, pREV and pMDL. HEK293T cells were cultured in IMDM (Sigma, I3390) supplemented with 10% FBS (Fisher Scientific, 11550356) and 5% pen-strep antibiotic (Sigma, P4458). 48 h after transfection, HEK293T conditioned medium was harvested, centrifuged and filtered using 0.22 µm filters. The viral p24 antigen concentration was measured using an HIV-1 p24 core profile enzyme-linked immunosorbent assay ELISA assay Lenti-X p24 Rapid Titer kit according to the manufacturer’s instructions. Serial dilutions of freshly harvested conditioned medium were used to infect 1.2 × 105 LN18 cells in a six-well plate in the presence of polybrene (8 μg ml−1). As a control, cells were transduced with pUltra-Chili lentiviral vector, containing TdTomato, but no IDH1 sequences.
Pen strep antibiotic
Manufactured by Merck Group
Sourced in United States
Pen/Strep antibiotics are a combination of penicillin and streptomycin, two commonly used antibiotics. They are used to inhibit the growth and reproduction of a wide range of bacteria. Pen/Strep antibiotics work by interfering with the formation of the bacterial cell wall, which is essential for the survival and proliferation of the microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Pbssvd2005, by Addgene (2 mentions)
Iscove modified dulbecco media (imdm), by Merck Group (1 mentions)
Exoquick tc kit, by System Biosciences (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Methyl α d mannopyranoside, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Trypsin solution, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Cytosine arabinoside, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Streptomycin sulfate salt, by Merck Group (1 mentions)
F12 nut mix, by Thermo Fisher Scientific (1 mentions)
Recombinant mouse ifn γ, by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Merck Group (1 mentions)
14 protocols using pen strep antibiotic
1
Lentiviral Transduction of IDH1 Mutants
2
Isolation of HEK-293T-derived Exosomes
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HEK-293T cells were cultured in 25 cm2 flasks containing RPMI 1640 medium supplemented with 10% exosome-depleted bovine serum (FBS; System Bioscience) and 1% Pen-Strep antibiotic (Sigma). The cells were incubated at 37°C in 5% CO2 for 48 h. Then, 24 h after cell passage (the cell confluency was 70–80%), the culture medium was slowly collected. Finally, HEK-293T-derived exosomes were isolated using ExoQuick-TC kit (System Biosciences). According to ExoQuick-TC protocol, the collected medium was centrifuged at 3000 g for 15 min to remove cells and debris. In the next step, 1 ml of ExoQuick-TC buffer was mixed with 5 ml of culture medium, and incubated at 4°C for 24 h. The mixture was centrifuged at 3000 g for 10 min to precipitate exosomes. The Bradford test was performed to measure protein levels at this stage. After that, the pellet was resuspended in various buffers (based on kit), and finally, the solution containing pure exosome was obtained. The BCA protein assay kit (Thermo Fisher Scientific) was used to measure protein content at this stage.
3
HeLa Cell Adhesion Assay
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HeLa cells were always kept at 37 °C in an atmosphere of 5% CO2, in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma), containing 10% fetal bovine serum (FBS) (Sigma) and 1% PenStrep antibiotic (10,000 U penicillin + 10 mg streptomycin/mL) (Sigma). The adhesion tests were performed according to Cravioto et al. [18 (link)]. Briefly, 1 mL of a suspension of HeLa cells at a concentration of 1 × 105 cells/mL (Neubauer chamber) was distributed in a 24-well microplate containing glass coverslips, incubated at 37 °C in an atmosphere of 5% CO2 for 48 h, until reaching the semi-confluence stage (70 to 90%). A 20-µL aliquot of each isolate (cultured overnight in BHI broth at 35 °C, without agitation) was added in duplicate to the microplate wells, already containing HeLa cells in DMEM supplemented with 2% FBS and 2% methyl α-D-mannopyranoside (Sigma). After 6 h of incubation, each well was washed six times with sterile PBS, and the slides were fixed with methanol (overnight), stained with May–Grünwald (5 min) and Giemsa (20 min), and analyzed under an oil immersion microscope. Isolates EPEC-E2348/69 (AL standard), EAEC-042 (AA standard), DAEC-C1845 (AD standard) and E. coli HB101 (NA standard) were used as controls [19 (link)].
4
Rat Choroid Plexus Cell Culture Protocol
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CP tissues were excised from the lateral ventricles of
neonatal rats (4-5 day-old), rinsed by phosphate-buffered
saline (PBS, Sigma, USA), and next, the incubation with
0.25% trypsin solution (Invitrogen-Gibco, England) was
performed for 20 minutes at 37˚C. The next step was the
addition of fetal bovine serum (FBS, Invitrogen-Gibco,
England) and the centrifuge for 5 minutes. The sediment
was transmitted to a culture medium, including Dulbeccoʼs Modified Eagleʼs Media (DMEM/F12, Invitrogen-gibco,
England), 10% FBS, and 1% pen/strep antibiotic (Sigma,
USA). 20 μM cytosine arabinoside (Sigma, USA) was
used to prevent fibroblast proliferation for one week. The
culture medium was changed every 48 hours (22 (link)).
neonatal rats (4-5 day-old), rinsed by phosphate-buffered
saline (PBS, Sigma, USA), and next, the incubation with
0.25% trypsin solution (Invitrogen-Gibco, England) was
performed for 20 minutes at 37˚C. The next step was the
addition of fetal bovine serum (FBS, Invitrogen-Gibco,
England) and the centrifuge for 5 minutes. The sediment
was transmitted to a culture medium, including Dulbeccoʼs Modified Eagleʼs Media (DMEM/F12, Invitrogen-gibco,
England), 10% FBS, and 1% pen/strep antibiotic (Sigma,
USA). 20 μM cytosine arabinoside (Sigma, USA) was
used to prevent fibroblast proliferation for one week. The
culture medium was changed every 48 hours (22 (link)).
5
Culturing A549 NSCLC Cells
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The NSCLC cell line A549 was purchased from the ATCC collection. Since cells were maintained in our laboratory, cell identification of A549 cells was performed by Microsynth AG (Balgach, Switzerland). Cells were maintained in DMEM/F12 (DMEM, PANTM Biotech; F12 Nut mix, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 4.5 g/L glucose, 10% fetal bovine serum (FBS, Gibco), 1X GlutaMAX (Gibco) 1% PenStrep antibiotics (Penicillin G sodium salt, and Streptomycin sulfate salt, Sigma-Aldrich, St. Louis, MI, USA). The cells were cultured in standard tissue culture Petri-dish (2D monolayer, 10 mm diameter dish, Corning Life Sciences, Corning, NY, USA); T-75 flasks (2D TC, tissue culture flasks T75 flask, TPP, Trasadingen, Switzerland) or collagen type I covered T-75 flasks (2D Coll, tissue culture flasks T75 flask, TPP) for time points day 4 and 9 at maximum 80% confluence at standard atmosphere of 95% air and 5% CO2 (Sanyo, Osaka, Japan). At about 80% confluence cells were washed, harvested with trypsin (Sigma-Aldrich) and seeded into new dish.
6
Bone Marrow Derived Macrophage Stimulation
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Extracted bone marrow cells were plated at density of 7 × 106 cells per 10 cm bacteriological dish and cultured for 7 days in RPMI (Sigma) medium supplemented with Pen-Strep antibiotics (Sigma), 10% FCS (Gibco), 50 μM β-mercaptoethanol (Gibco), GlutaMaxTM (Invitrogen) and 20 ng mL−1 of murine GM-CSF (Peprotech, #315-03). The medium was topped up after 4 days of culture. On day 8, BMDMs were plated in round bottomed 96-well plates at density of 1.5 × 105 cells/well and the following day the BMDMs were treated with various microbial stimuli in the presence of 5 ng mL−1 recombinant mouse IFNγ (Peprotech, #315-05) for 3 h, 8 h, or 24 h. Hh (defrosted bacterial pellets) was used at 0.5 OD/well, ultrapure LPS from E. coli O111:B4 at 10 ng mL−1 (#tlrl-3pelps), Pam3CSK4 (#tlrl-pms)—100 ng mL−1, zymozan (TLR2/dectin-1 agonist, #tlrl-zyn)—1 μg mL−1, ODN1826—0.2 μg mL−1 (CpG, TLR9 agonist, #tlrl-1826), HKMT—10 μg mL−1 (Mincle agonist, #tlrl-hkmt-1), neutralising anti-mouse TLR2 antibody (clone C9A12, # mabg-mtlr2)—0.3 μg mL−1 (all from Invivogen). Cytokine expression in the culture supernatant was determined using mouse IL-12 Duoset ELISA kit (RnD Systems) and mouse IL-23 ELISA Ready-SET-Go!™ Kit (eBioscience).
7
In Vitro Prostate Cancer Cell Culture
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For the in vitro drug perturbation validations, we used the androgen-sensitive prostate adenocarcinoma cell line LNCaP purchased from American Type Culture Collection (ATCC, Manassas, WV, USA). ATCC found no Mycoplasma contamination and the cell line was identified using STR profiling. Cells were maintained in RPMI-1640 culture media (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 4.5 g/L glucose, 10% foetal bovine serum (FBS, Gibco), 1 X GlutaMAX (Gibco), 1% PenStrep antibiotics (Penicillin G sodium salt, and Streptomycin sulfate salt, Sigma-Aldrich, St. Louis, MI, USA). Cells were maintained in a humidified incubator at 37 °C with 5% CO2 (Sanyo, Osaka, Japan).
8
SV40-Immortalized MEF Cell Culture
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E14.5 embryos were dissected from uterus; head, limbs and internal organs were removed. Tissue was disaggregated using an 18-gauge syringe and brought to single cell suspension with trypsin incubation at 37°C. Cells were then plated onto 100 mm tissue culture plates, passaged upon confluency and maintained in Dulbecco's modified Eagle medium (DMEM-Sigma) with 10% fetal bovine serum (FBS-Gibco) and 1X Pen/Strep antibiotics (Sigma). SV40 T immortalized MEFs (iMEFs) were derived from primary Hat1+/+ and Hat1−/− embryonic day 13.5 embryos. To establish iMEFs, early passage cells were transformed with SV-40 T antigen containing plasmid pBSSVD2005 (ADDGENE, Cambridge, MA). Early passage cells were seeded at 25% confluency in six-well plates and transfected with 2 ug of expression vector using Fugene reagent (Roche). Cells were harvested and seeded into 100 mm dishes after 48 h of transfection. The cells were split at 1 in 10 dilutions until passage 5.
For the SILAC experiments, cells were grown in SILAC DMEM media (ThermoFisher Scientific) and supplemented with 10% dialyzed fetal bovine serum (ThermoFisher Scientific), antibiotics and isotopically labeled lysine and arginine (Cambridge Isotope laboratories). Hat1−/− cells were grown under these conditions for at least four passages to guarantee the full incorporation of the labeled amino acids.
For the SILAC experiments, cells were grown in SILAC DMEM media (ThermoFisher Scientific) and supplemented with 10% dialyzed fetal bovine serum (ThermoFisher Scientific), antibiotics and isotopically labeled lysine and arginine (Cambridge Isotope laboratories). Hat1−/− cells were grown under these conditions for at least four passages to guarantee the full incorporation of the labeled amino acids.
9
BHK-21 Cell Culture Protocol
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Baby hamster kidney 21 (BHK) (ATCC ® CCL-10™) cells were grown in cell culture flasks in an incubator at 37°C with 95% relative air humidity and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza, Verviers, Belgium) with 10% foetal bovine serum (FBS) (Delta products, Johannesburg, South Africa), 1% L-Glutamine (L-Glut) (Sigma Aldrich, Ayrshire, United Kingdom), 1% non-essential amino acids (NEAA) (Lonza, Verviers, Belgium), and 1% penicillin/streptomycin (Pen/Strep) antibiotics (Sigma Aldrich, Ayrshire, United Kingdom). According to their doubling time, cells were passaged every 3–5 days to keep it in the logarithmic growth phase. For passaging, the culture media was removed, and the cells were washed with 1 x phosphate buffered saline (PBS).
10
Colon Explant Culture for IFNγ
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3 mm segments isolated from a middle part of mouse colon were cultured overnight in RPMI media supplemented with Pen-strep antibiotics (Sigma), 10% FCS (Gibco) and 50 μM β-mercaptoethanol (Gibco). IFNγ was quantified in the supernatant by enzyme-linked immunosorbent assay (ELISA, R&D Systems, UK) and normalized to explant weight (mg of tissue).
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