BT-474 and BT-549 cells were cultured in RPMI-1640 (Wisent, 350-000-CL) media supplemented with 10% fetal bovine serum (FBS) (Wisent, 080–150), 1% penicillin-streptomycin (Penstrep) (Wisent, 450-200-EL), 10μg/ml insulin (Wisent 521–016). MDA-MB-468, MDA-MB-231, MDA-MB-453, SUM-149 and SUM-159 cells were maintained in RPMI-1640 media supplemented with 10% FBS and 1% Penstrep. BT-20 cells were maintained in RPMI-1640 media supplemented with 10% FBS, 1% Penstrep, 2mM L-glutamine (Wisent 609–065) and 1mM sodium pyruvate (Wisent 600–110). SKBR3 cells were maintained in McCoy’s 5a (Wisent, 217-010-XK) media supplemented with 10% FBS and 1% Penstrep. MCF-7 cells were maintained in phenol red-free DMEM F12 (Wisent 319–080) supplemented with 5% FBS, 1% Penstrep, 13.4 ml sodium bicarbonate 7.5% solution (Wisent 609–105), 7.5 ml HEPES 1M (Wisent 330–050) and 50μl estradiol 10-5M (E2) (Sigma E8875). ZR-75-1 cells were maintained in RPMI-1640 media supplemented with 10% FBS, 1% Penstrep, 2mM L-glutamine, 7.5 ml HEPES, 13.4 ml sodium bicarbonate and 50μl E2. JIMT-1 cells were maintained with DMEM (Wisent, 319-005-CL) media supplemented with 10% FBS and 2mM L-glutamine. Cell lines were grown at 37°C with 5% CO2 and tested for mycoplasma infections (Lonza, MycoAlert PLUS). Cells were cultured to obtain 60–80 million cells, pelleted and suspended in formalin + 3% agar and FFPE.
Pen strep
Manufactured by Wisent
Sourced in Canada
Pen/Strep is a sterile liquid solution containing penicillin and streptomycin. It is commonly used as an antibiotic supplement in cell culture media to prevent bacterial contamination.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Wisent (5 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Pen strep, by Merck Group (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Insulin, by Roche (2 mentions)
Rpmi media, by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Htert rpe1, by American Type Culture Collection (2 mentions)
Mccoy s 5a modified medium, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (2 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
Insulin, by Merck Group (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
L glutamine, by Wisent (2 mentions)
Hi fbs, by Wisent (2 mentions)
Rbc lysis buffer, by BioLegend (2 mentions)
L glutamine, by Merck Group (2 mentions)
Rpmi 1640, by Wisent (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
16 protocols using pen strep
1
Breast Cancer Cell Line Cultivation and Preservation
2
Cell Culture of Mammary Cell Lines
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MCF-12A, MCF-10A and MCF-7 were purchased from the ATCC. MCF-12A and MCF-10A cell lines were maintained in DMEM/F12 (Thermo Fisher Scientific) supplemented with 5% horse serum (Invitrogen), 20 ng/mL EGF (Invitrogen), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 μg/mL insulin (Roche) and 1X pen/strep (Wisent). The MCF-7 cell line was maintained in DMEM/F12 with 10% fetal bovine serum (Wisent) and 1X pen/strep.
3
Cell Culture Conditions for Various Cell Lines
4
Cell Culture Protocols for Multiple Cell Lines
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RPE1-hTERT, HCT116, and 293T cells were purchased from American Type Culture Collection. HeLa T-Rex Flp-In cells orginated from the laboratory of Stephen Taylor and were a kind gift from Laurence Pelletier. RPE1-hTERT-derived cell lines, 293T-derived cell lines, and HeLa T-Rex Flp-In-derived cell lines were grown in Dulbecco's modified Eagle medium (DMEM) (Gibco/Thermo Fisher, 11965092) supplemented with 10% fetal bovine serum (FBS) (Wisent), 1× GlutaMAX, 1× nonessential amino acids (both Gibco/Thermo Fisher), and 100 U/mL penicillin and 100 µg/mL streptomycin (Pen/Strep) (Wisent). SUM149PT Cas9 cell origin and culture conditions were as described previously (Zimmermann et al. 2018 (link)). HCT116-derived cell lines were grown in McCoy's 5A (Gibco, 1660-082) + 10% FBS (Gibco) + 1% penicilllin–streptomycin mix (Nakalai Tesque, 09367-34) + 1× L-glutamine (final 2 mM; Wako Chemical, 073-05391). RPE1-hTERT Cas9 TP53-KO BRCA1-KO were cultured at 37°C and 3% O2. All other cell lines were grown at 37°C and atmospheric O2. All cell lines were routinely authenticated by STR and tested negative for mycoplasma.
5
Cell Culture Conditions for Cancer Research
6
Visualizing Murine Glioblastoma Cells
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Murine-derived glioblastoma cell line stably expressing the luciferase reporter protein (GL261-Luc) was purchased from PerkinElmer (Waltham, MA, USA, catalog number BW134246) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Scientific, Waltham, MA, USA) supplemented with 10% FBS and 1% PenStrep (Wisent Inc, Saint-Jean Baptiste, QC, Canada). For microscopy studies, 50 k GL261 cells were seeded in a coverslip-containing 24-well plate 12 h prior to the experiment for adherence. The following day, HA-LNPs 3× or 5× were added to the cells at the desired final concentration. After overnight incubation, cells were fixed with paraformaldehyde 4%, washed thrice with PBS, and stained with Hoechst 33,342 at 2 μg mL−1 for 30 min, followed by PBS washing and mounting over glass slides for imaging with a Leica TCS SP8 microscope (Wetzlar, Germany). Pictures were analyzed on Fiji software (version 2.9.0/1.53t).
7
CFTR Functional Assay in 16HBEge Cells
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16HBE14o- cells, previously genome-edited to produce the homozygous CFF-16HBEge W1282X-CFTR cell line, were obtained from the Cystic Fibrosis Foundation.44 (link) Cells were grown at 37°C for 5 days post-confluence, submerged on 96-well black-well, clear-bottom culture plates (Costar) in EMEM media (Wisent BioProducts) with 10% fetal bovine serum (Wisent BioProducts) and 1% Pen-Strep (Wisent BioProducts). 24 h before the assay, cells were treated with DMSO, 3 μM VX-809 (Selleck Chemicals), and 10 μM VX-661 (Selleck Chemicals) + [R]-VX-445 (MedChemExpress). Cells were then loaded with blue, membrane potential dye dissolved in chloride-free buffer (150 mM NMDG-gluconate, 3 mM potassium gluconate, 10 mM HEPES, pH 7.30, 300 mOsm) for 30 min. The plate was then read in a FLIPR Tetra (Molecular Devices) at 37°C (excitation: 530 nm, emission: 560 nm). CFTR was stimulated with 10 μM forskolin (Sigma-Aldrich) and either 1 μM VX-770 (Selleck Chemicals) or DMSO. The assay was terminated with 10 μM CFTRinh172 (Cystic Fibrosis Foundation Therapeutics). Changes in membrane potential were normalized to the point before the addition of agonist and to the DMSO control response.82 (link),83 (link)
8
Cell Culture Conditions for Various Cell Lines
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HeLa, RPE1-hTERT and 293T cells were purchased from ATCC and grown in Dulbecco’s Modified Eagle Medium (DMEM; Gibco/Thermo Fisher) supplemented with 10% fetal bovine serum (FBS; Wisent), 200 mM GlutaMAX, 1x non-essential amino acids (both Gibco/Thermo Fisher), 100U/ml penicillin and 100μg/ml streptomycin (Pen/Strep; Wisent). HCT116 TP53-KO cells34 (link), a kind gift from B. Vogelstein (Johns Hopkins University School of Medicine), were maintained in modified McCoy’s 5A medium (Gibco/Thermo Fisher) supplemented with 10% FBS and Pen/Strep. SUM149PT cells were purchased from Asterand BioScience and grown in a DMEM:F12 medium mixture (Gibco/Thermo Fisher) supplemented with 5% FBS, Pen/Strep, 1 μg/ml hydrocortisone and 5 μg/ml insulin (both Sigma). DLD-1 WT and BRCA2-KO cells were purchased from Horizon and maintained in RPMI media (Gibco/Thermo Fisher) supplemented with 10% FBS and Pen/Strep. All cell lines were grown at 37°C and 5% CO2. HeLa, RPE1-hTERT (with the exception of BRCA1-KO and POLBΔ188-190 clones) and HCT116 cells were grown at atmospheric O2. RPE1-hTERT BRCA1-KO and POLBΔ188-190 clones, as well as DLD-1 and SUM149PT cell lines were maintained at 3% O2.
9
Culturing MCF-10A and MCF-12A Cells in Matrigel
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MCF-10A and MCF-12A cells were cultured in Matrigel following an adapted protocol from Debnath et al.24 (link). Briefly, 8-well chamber permanox slide (Thermo Scientific) was coated with Growth Factor Reduced Matrigel Matrix, (Matrigel), (BD Biosciences). 5000 MCF-12A cells were seeded in DMEM/F12 phenol red free media supplemented with 2% charcoal stripped horse serum (Invitrogen), 5 ng/mL EGF (Invitrogen), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 μg/mL insulin (Roche), 1X pen/strep (Wisent) and 2% Matrigel. MCF-12A cells were treated with BPA or BPS (0.1, 1, and 10 μM) or 1 nM oestrogen (E2) (Sigma) or 0.1% ethanol. The treatment media was changed every 4 days and the cells were grown until time points at days 8, 16 and 25. For the experiments using ICI 182,780 (Sigma), the cells were plated in Matrigel as described above in the presence or absence of 1 µM ICI 182,780.
10
Clonal Isolation of Edited Cells
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FACS was used to enrich populations of edited cells or for clonal isolation. Cells were sorted using a FACSMelody cell sorter (BD Biosciences) after recovery from antibiotic selection or 8 days after nucleofection for endogenous tagging. Briefly, cells were dissociated using Accutase, resuspended in PBS (Wisent), and then passed through a 35 μm strainer to remove large cell clumps. Cells were sorted using gates set to capture individual fluorescent cells. Individual cells or enriched populations of 500 tagged cells were sorted into individual wells of a 96-well plate containing recovery media [mTeSR Plus media with CloneR2 reagent and Pen-Strep (50 units/mL Penicillin and 50 μg/mL Streptomycin; Wisent)]. To recover clones, cells were kept in recovery media for 6 days with media changes on days 2 and 4. The media was then changed daily with mTeSR Plus until day 11. On day 11, the cells were passaged into fresh 96-well plates and grown to confluency before freezing and screening.
For flow cytometry, cells were prepared and analyzed using the same protocol, and data was analyzed using the R package CytoExploreR (Hammill, 2021 ).
For flow cytometry, cells were prepared and analyzed using the same protocol, and data was analyzed using the R package CytoExploreR (Hammill, 2021 ).
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