HEK293 (ATCC), HeLa (ATCC), and SLK cells were maintained in DMEM (Thermo Fisher) containing 10% FBS (VWR), 1% Pen-Strep (Thermo Fisher), and 1% l -glutamine (Thermo Fisher). 293TT cells (a kind gift from Dr. Cary Moody) were maintained in in DMEM containing 10% FBS (Sigma-Aldrich) and 1% Pen-Strep. iSLK.219 cells were maintained in DMEM containing 10% Tet-free FBS (Clontech), 1% Pen-Strep, 1% l -glutamine, 10 μg/mL puromycin (Corning), 250 μg/mL Geneticin (Thermo Fisher), and 400 μg/mL hygromycin B (Corning). BCBL1-TREx-RTA cells (a kind gift from Dr. Jae Jung) were maintained in RPMI 1640 (Corning) containing 10% Tet-free FBS (Clontech), 1% Pen-Strep, 1% l -glutamine, 1% sodium bicarbonate (Thermo Fisher), 0.05 mM β-mercaptoethanol (Thermo Fisher), and 200 μg/mL hygromycin. AGS-EBV cells (a kind gift from Dr. Lindsey Hutt-Fletcher) were maintained in F-12 media (Thermo Fisher) containing 10% FBS (VWR), 1% Pen-Strep, 1% l -glutamine, and 500 μg/mL Geneticin. Akata-BX1 cells (a kind gift from Dr. Lindsey Hutt-Fletcher) were maintained in RPMI 1640 containing 10% FBS (VWR), 1% Pen-Strep, 1% l -glutamine, 0.05 mM mercaptoethanol, and 500 μg/mL Geneticin. AGS cells (ATCC) were maintained in F-12 media containing 10% FBS (VWR), 1% Pen-Strep, and 1% l -glutamine. All cells were maintained at 37 °C and 5% CO2 in a regularly cleaned and decontaminated laboratory incubator.
Pen strep
Manufactured by Avantor
Sourced in Belgium
Pen-Strep is a liquid solution containing a combination of penicillin and streptomycin. It is a commonly used antibiotic supplement for cell culture media.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (9 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Pen strep, by Merck Group (3 mentions)
Pen strep, by Thermo Fisher Scientific (3 mentions)
Collagenase, by Thermo Fisher Scientific (2 mentions)
Laminin, by Merck Group (2 mentions)
Dispase, by Thermo Fisher Scientific (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Dulbecco modified eagle medium (dmem), by HiMedia (2 mentions)
Human epidermal growth factor (hegf), by STEMCELL (2 mentions)
Insulin, by Thermo Fisher Scientific (2 mentions)
Bovine pituitary extract, by Merck Group (2 mentions)
Mcf10a cells, by American Type Culture Collection (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
Cholera toxin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Atlas Biologicals (2 mentions)
L glutamine, by Corning (2 mentions)
Fetal bovine serum (fbs), by Gemini Bio (2 mentions)
Mda mb 231 tnbc cells, by American Type Culture Collection (2 mentions)
13 protocols using pen strep
1
Cell Culture Conditions and Maintenance
2
Isolation and Culture of Mouse DRG Neurons
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DRGs were isolated from mice and dissociated in 1ml of media containing 2.5U/ml of dispase (Fisher) and 2.5mg/ml of collagenase (Fisher). After dissociation, the cells were washed with complete media (DMEM (HiMedia) with 10% FBS (Atlanta Biologicals) and 1% PenStrep (VWR)) and pelleted at 1000 rpm for 15 minutes. Approximately 30μl of the cell suspension was plated on 18mm round glass slides with a coating of laminin (Sigma Aldrich) and poly-L-lysine (Sigma Aldrich) and incubated for 1.5 hours. Afterward, 1ml of complete media was added, and the cells were incubated overnight. All incubation steps were done at 37°C with 5%CO2.
3
Culturing MDA-MB-231 and MCF-10A Cell Lines
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MDA-MB-231 TNBC cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, VWR International) supplemented with 10% fetal bovine serum (FBS; Gemini Bio Products) and 1% penicillin-streptomycin (pen-strep; VWR International). MCF-10A cells (ATCC) were cultured in DMEM supplemented with 1% pen-strep, 5% FBS, 50 μg/mL bovine pituitary extract (Sigma), 0.5 μg/mL hydrocortisone (Sigma), 20 ng/mL human epidermal growth factor (STEMCELL Technologies), 10 μg/mL insulin (Thermo Fisher Scientific), and 100 ng/mL cholera toxin (Sigma). The cultures were maintained at 37°C in a 5% CO2 humidified environment. When cells reached 80%–90% confluency in T75 cell culture flasks, they were passaged or plated by detaching the cells from the flask using trypsin-EDTA (Thermo Fisher Scientific) and then counting cells with a hemocytometer.
4
GDA and Compound 5 Synergistic Effects
5
Culturing HUVECs and MC3T3-E1 Cells
6
In Vitro Endothelial Cell Culture
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In vitro experiments were performed with mouse aortic endothelial cells (iMAECs, provided by H. Jo at Emory) (29 ), HEK cells (HEK293, GenTarget), iMAECs stably transduced with CAG-SpCas9–enhanced GFP, produced in the Dahlman Lab, or mouse aortic endothelial cells (C57E κB-GFP) (provided by M. Schwartz at Yale). In all cases, cells were maintained at 37°C and 5% CO2 and cultured using previously established conditions. In all cases, cell media was supplemented by penicillin-streptomycin [penicillin G (500 U/ml) and streptomycin (0.5 mg/ml)] (PenStrep, VWR) and 10% (v/v) fetal bovine serum (FBS; VWR). HEKs were passaged with Dulbecco’s modified Eagle’s medium (DMEM) F-12 50/50 (Corning). iMAECs were passaged using DMEM with glucose (1 g/liter), l -glutamine, and sodium pyruvate (Corning), supplemented by 1% (v/v) MEM nonessential amino acid solution (MEMNEAA, Sigma-Aldrich) and endothelial cell growth supplement (25 μg/ml; EMD Millipore). C57E κB-GFP was passaged with EGM-2 media (Lonza).
Unless otherwise noted, cells were seeded in a 24-well plate at a density of 40,000 cells per well with 500 μl of media per well. Twenty-four hours later, LNPs were added with a total RNA dose of 50, 150, or 250 ng per well. Initial experiments done with L2K used a total RNA dose of 400 ng per well.
Unless otherwise noted, cells were seeded in a 24-well plate at a density of 40,000 cells per well with 500 μl of media per well. Twenty-four hours later, LNPs were added with a total RNA dose of 50, 150, or 250 ng per well. Initial experiments done with L2K used a total RNA dose of 400 ng per well.
7
Culturing Human Embryonic Kidney Cells
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This study has used the following cell lines: HEK293T cells purchased from the American Type Culture Collection (ATCC; Cat#: CRL-3216), Expi293F cells from ThermoFisher Scientific (Cat#: R79007) and 293T/17 cells from the ATCC (Cat#: CRL-11268). All cells were grown following the standard protocols recommended by the manufacturer. Briefly, they were grown in DMEM (Gibco Cat: #10313), Pen Strep (Gibco Cat#: 15140-122), GlutaMAX (Gibco Cat#: 35050-061), and 10%FBS (Avantor/Seradigm Cat#:97068-085) at 37°C with 5.5% CO2 in T-Flasks or plates, or for suspension culture, in Expi 293 Expression Medium (Gibco Cat#:A14351) and Pen Strep in VWR Erlenmeyer Flasks at 37°C with 8% CO2, 80% humidity and 130 rpm in a shaker incubator.
8
Isolation and Culture of Mouse DRG Neurons
9
Culture and Passage of TNBC and Normal Breast Cell Lines
10
Culturing MDA-MB-231 TNBC Cells
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MDA-MB-231 human TNBC cells (American
Type Culture Collection, ATCC) were cultured in Dulbecco’s
modified Eagle’s medium (DMEM; Fisher Scientific) supplemented
with 10% fetal bovine serum (FBS; Gemini Bio Products) and 1% penicillin–streptomycin
(pen-strep; VWR). Cells were cultured in T75 cell culture flasks at
37 °C in a 5% CO2 environment. Cells were passaged
or plated when they reached 80–90% confluency and were detached
from the flask using 3 mL of 0.25% trypsin–EDTA (ThermoFisher).
Cells were counted using a hemocytometer.
Type Culture Collection, ATCC) were cultured in Dulbecco’s
modified Eagle’s medium (DMEM; Fisher Scientific) supplemented
with 10% fetal bovine serum (FBS; Gemini Bio Products) and 1% penicillin–streptomycin
(pen-strep; VWR). Cells were cultured in T75 cell culture flasks at
37 °C in a 5% CO2 environment. Cells were passaged
or plated when they reached 80–90% confluency and were detached
from the flask using 3 mL of 0.25% trypsin–EDTA (ThermoFisher).
Cells were counted using a hemocytometer.
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