The human colorectal adenocarcinoma LoVo, SW480 and DLD-1 cell lines were gifted by Eok-Soo Oh (Ewha Womans University). LoVo cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI-1640; #31800022; Gibco, Grand Island, NY, USA), SW480 cells were maintained in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM/F-12; #12500062; Gibco) and DLD-1 cells were maintained in DMEM (#12100046; Gibco) supplemented with 10 % heat-inactivated fetal bovine serum (FBS; #US-FBS-500; GW Vitek, Seoul, South Korea), 100 units/ml penicillin and 100 µg/ml streptomycin (#LS202-02; WelGENE, Daegu, South Korea). In addition, HEK293T cells were maintained in DMEM supplemented with 10 % FBS (#US-FBS-500; GW Vitek), 100 units/ml penicillin and 100 ug/ml streptomycin (#LS202-02; WelGENE). The cells were incubated at 37 °C in a humidified incubator with 5 % CO2. For transient transfection, 1–3 µg of plasmids was transfected into HEK293T cells using the calcium phosphate precipitation method. SW480 cells were transfected using Lipofectamine 3000 Reagent (#L3000015; Invitrogen) according to the manufacturer’s instructions.
Ls202 02
Manufactured by Welgene
Sourced in United States
The LS202-02 is a laboratory centrifuge designed for general-purpose applications. It features a maximum speed of 6,000 RPM and can accommodate rotors with a capacity of up to 4 x 50 mL. The centrifuge is compact and suitable for use in a range of laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Lm001 05, by Welgene (5 mentions)
Streptomycin, by Welgene (2 mentions)
Penicillin, by Welgene (2 mentions)
Pk004 07, by Welgene (2 mentions)
S001 07, by Welgene (2 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Hepes, by Welgene (1 mentions)
Fetal bovine serum (fbs), by Welgene (1 mentions)
Rpmi 1640, by Welgene (1 mentions)
Fugene, by Promega (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by Merck Group (1 mentions)
B16f10 cells, by Thermo Fisher Scientific (1 mentions)
M254500, by Thermo Fisher Scientific (1 mentions)
Nheks, by Thermo Fisher Scientific (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Nhems, by Thermo Fisher Scientific (1 mentions)
Control sirna, by Bioneer (1 mentions)
11 protocols using ls202 02
1
Culturing and Transfecting Human Colorectal Cancer Cell Lines
2
Gastric Cancer Cell Line Cultivation
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Human gastric cancer cell lines, MKN28 and AGS, were acquired from the Korean Cell Line Bank. The cells were used within ten passages. All cell lines were cultured in RPMI1640 (WelGENE, Gyeongsangbuk-do, Republic of Korea, LM011-07) with 25 mmol/L HEPES (WelGENE, BB001-01), 10% FBS (WelGENE), 100 U/mL penicillin (WelGENE, LS202-02), and 100 mg/mL streptomycin (WelGENE, LS202-02). Cells were incubated at 37 °C with 5% CO2. Transfection agent FuGENE (Promega, Wisconsin, USA, E5911) was used to facilitate transfection.
3
TGF-β1 Activation of NLRP3 Inflammasome in LX-2 HSCs
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LX-2 cells (SCC065, Millipore, MA, USA), a human HSC line, were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM; LM001-70, WelGene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS; PK004-07, WelGene) and 1% penicillin–streptomycin (P/S; LS202-02, WelGene). Cultured LX-2 cells were starved in 1% FBS-DMEM for 24 h and activated by treatment with 10 ng/mL TGF-β1 (100–21, PEPROTECH, NJ, USA) for 24 h. For the NLRP3 inflammasome activation study, cultured LX-2 cells were starved with serum-free DMEM for 24 h and treated with 10 ng/mL TGF-β1 for 10, 30, and 60 min. Non-treated cells were used as the control. After each incubation time, LX-2 cells were harvested, cultured media were used for ELISA analysis, and the cells were used for protein expression and mRNA expression analysis. The experiment using the TAK1 inhibitor and IL-1R antagonist proceeded as follows. LX-2 cells were untreated (-) or pre-treated with 50 nM of (5Z)-7-oxozeanol or 1 μg/ml of IL-1R antagonist (SRP3327, Sigma) for 1 h and then treated with 10 ng/ml of TGF-β1 for 10 min in the presence of an inhibitor or antagonist. Cell lysates and culture media were prepared for western blotting and ELISA.
4
Cell Culture and LTAP Exposure Protocol
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Human HaCaT cells, HDFs, NHEKs, NHEMs and murine B16F10 cells were purchased from Thermo Fisher Scientific (Waltham, MA, USA). HaCaT cells, HDFs and B16F10 cells were cultured in Dulbecco’s modified Eagle’s medium (HyClone, GE Healthcare Life Sciences, Logan, UT, USA) with 10% fetal bovine serum (HyClone) and 1% penicillin–streptomycin (LS202-02, Welgene, Daegu, Korea). NHEKs and NHEMs were cultured in keratinocyte media (MEP1500CA, Gibco, Life Technologies, Carlsbad, CA, USA) and Medium 254 (M254500, Gibco) supplemented with human keratinocyte and melanocyte growth supplement (S0015, S0025, Gibco) and 1% penicillin-streptomycin at 37 °C in a humidified mixture of air (95% (v/v)) and 5% CO2. For LTAP exposure, 5 × 105 cells were seeded in 35 mm dishes and incubated for 24 h. Before LTAP exposure, the medium was exchanged for 2 mL of new growth medium. The lid was opened, and the dish was placed 1 cm below the part where argon gas was ejected from the LTAP generator, and exposed for 1, 3, or 5 min. After exposure for the indicated time, the dishes were placed in a CO2 incubator at 37 °C.
5
Culturing and Manipulating HT22 Mouse Hippocampal Cells
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HT22 mouse hippocampal cells were cultured as previously described20 (link),38 (link). HT22 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; LM-001–05; Welgene, Gyeongsan, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS; FB02–500; Serum Source, NC, USA) and antibiotics (penicillin and streptomycin; LS-202–02; Welgene, Gyeongsan, Korea) at 37oC in a humidified incubator with 95% air and 5% CO2.
Cells grown to 70–80% confluence in a 6-well plate or a 100-mm dish were treated with Corticosterone, or drugs in DMEM containing 1% FBS. After 24 h, cells were washed in 1X phosphate buffered saline (PBS) and harvested. Corticosterone, RU486, AICAR, or Compound C were applied at the dose indicated. Corticosterone and RU486 were purchased from Tocris.
Cells were transfected with siRNA using Lipofectamine (Invitrogen, Carlsbad, CA, USA) as an reagent in DMEM containing 1% FBS according to the manufacturer’s protocol. The final concentration of siRNA was 100 nM, and the concentration of Lipofectamine-2000 was 7.5 μl/well.
The siRNAs used were: siRNA-control (SN-1012), siRNA-GR (#1393304, NM_008173.3), siRNA-Ppp2ca (#1411897, NM_019411.4), and siRNA-Creb (#1342686, NM_009952.2), which were obtained from Bioneer Co. (Daejeon, Korea).
Cells grown to 70–80% confluence in a 6-well plate or a 100-mm dish were treated with Corticosterone, or drugs in DMEM containing 1% FBS. After 24 h, cells were washed in 1X phosphate buffered saline (PBS) and harvested. Corticosterone, RU486, AICAR, or Compound C were applied at the dose indicated. Corticosterone and RU486 were purchased from Tocris.
Cells were transfected with siRNA using Lipofectamine (Invitrogen, Carlsbad, CA, USA) as an reagent in DMEM containing 1% FBS according to the manufacturer’s protocol. The final concentration of siRNA was 100 nM, and the concentration of Lipofectamine-2000 was 7.5 μl/well.
The siRNAs used were: siRNA-control (SN-1012), siRNA-GR (#1393304, NM_008173.3), siRNA-Ppp2ca (#1411897, NM_019411.4), and siRNA-Creb (#1342686, NM_009952.2), which were obtained from Bioneer Co. (Daejeon, Korea).
6
Culturing Human Colorectal Cancer Cell Lines
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The human colorectal cancer cell lines, SNU-C5, SW480, DLD-1, HCT-15, Caco-2, and SNU-C4, were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). The cells were cultured with RPMI 1640 medium (LM011-51, WELGENE, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS) (26140079, Gibco, Waltham, MA, USA) and 1% penicillin–streptomycin (LS202-02, WELGENE) solution. SW48 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured with DMEM medium (11995065, Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin. Cetuximab-resistant cells were maintained in a medium with 400 μg/mL cetuximab. All the cells were maintained every 3–4 days and cultured at 37 ℃ in a humidified 5% CO2 incubator.
7
Regulating Autophagy in NIH/3T3 Cells
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NIH/3T3 mouse embryonic fibroblasts were obtained from the Korean Cell Line Bank and cultured in DMEM (LM001-05, Welgene) supplemented with 10% FBS (fetal bovine serum; 26140-079, Gibco) and 1% penicillin–streptomycin (LS 202-02, Welgene). Under normal conditions, NIH/3T3 cells were cultured in 10% FBS, whilst under serum starvation conditions they were cultured in 0.5% FBS for 24 h. All cells were incubated in a humidified incubator with 5% CO2 at 37°C. To regulate autophagy in vitro, cells were treated with 5 nM of rapamycin (R8781, Sigma-Aldrich) and 50 uM of chloroquine (CQ; C6628, Sigma-Aldrich) for 4 h. Drugs were treated after 24 h serum starvation when it is necessary to induce ciliogenesis.
8
Culture and Maintenance of Renal Cell Lines
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Inner medullary collecting duct cells (IMCD cells; CRL-2123™, ATCC, Manassas, VI, USA) were cultured in DMEM/F12 medium (LM002-04, Welgene, Gyeongsan, Korea) containing 10% foetal bovine serum (FBS; 26140-079, Gibco, Waltham, MA, USA), and 1% penicillin-streptomycin (LS 202-02, Welgene). Normal human renal cortical epithelial cells (HRCE; CC-2554, LONZA, Morristown, NJ, USA) were cultured in REGM medium (CC-4190, LONZA) containing 1% penicillin-streptomycin. WT9-7 (CRL-2830™, ATCC), a proximal cortical tubule epithelial cell line isolated from renal cysts from a patient with ADPKD, was cultured in DMEM (LM001-05, Welgene) containing 10% FBS and 1% penicillin-streptomycin in culture dishes coated with type I bovine collagen (#354231, Corning, New York, NY, USA). Human embryonic kidney 293T cells (HEK293T) were cultured in DMEM (LM001-05, Welgene) containing 10% FBS and 1% penicillin-streptomycin in Poly-D-lysine-coated dishes (P7280, Sigma-Aldrich, St. Louis, MO, USA). These cells were grown in a humidified 5% CO2 incubator at 37°C. For hypoxic conditions, cells were incubated in 5% CO2 and 1% O2 balanced with N2 in a hypoxic chamber within an incubator at 37°C.
9
Establishing 4T1 Breast Cancer Cell Lines
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4T1 mouse breast cancer cells were obtained from the American Type Culture Collection (ATCC) (CRL-2539TM). 4T1-EpiKD cells, a subline of 4T1 cells in which epithin/PRSS14 was knocked down by stably incorporating shRNA, have been previously described (Kim et al., 2011 (link)). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (LM001-05; Welgene, Korea) supplemented with 10% fetal bovine serum (FBS) (S001-07; Welgene), 1X penicillin-streptomycin (LS202-02; Welgene), and 4 mM L-glutamine (LS002-01; Welgene). DMEM was replaced with RPMI1640 (RPMI, LM011-01; Welgene) or Iscove’s modified Dulbecco’s medium (IMDM) (LM004-01; Welgene) when needed. 4T1-I cells were generated by growing 4T1 cells in IMDM for 6 weeks. For transient transfection, Polyjet (SL100688; SignaGen, USA) was used according to the manufacturer’s instructions.
10
Isolation of Primary Murine Astrocytes
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To culture primary astrocytes, the brains of C57BL/6 mice were isolated at postnatal days 0–2. To obtain the cerebral cortex for use in the experiment, the cerebrum was separated, and the medulla was removed. The cerebral cortex was then pulverized by pipetting and cultured in a culture dish coated with 0.1 mg/mL Poly D-lysine (PDL, #354210, Corning). The culture medium for obtaining astrocytes was high-glucose Dulbecco’s modified Eagle medium (#LM001, Welgene) with 10% fetal bovine serum (#S001-07, Welgene), 10% horse serum (#26050-088, Gibco), and 1% penicillin/streptomycin (#LS202-02, Welgene) 05, Welgene). Primary astrocytes were incubated at 37°C and 5% CO2 for 3 days.
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