Syngeneic melanoma and lung carcinoma model were established on C57BL/6 male mice (aged 8 weeks) via subcutaneous inoculation of 1 × 106 B16F10 cells (CRL-6475, ATCC) or 2 × 106 LLC cells (CRL-1642, ATCC). Mice were randomly divided into four groups when tumor size reached 50 mm3 and AA (10 mg/kg), NG (50 mg/kg), or a combination of both was administered daily via i.p. injection as described previously.25 (link) Mice were harvested 4 weeks after the start of treatment or when tumor size reached 2,000 mm3 and then the invasive and metastatic nodules were counted. All animal experiments were carried out by a protocol approved by Animal Ethics Experimental Committee (AEEC) at the Chinese University of Hong Kong.
Crl 1642
Manufactured by American Type Culture Collection
Sourced in United States
The CRL-1642 is a cell line established from human breast adenocarcinoma tissue. It is a laboratory tool used for cell culture and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Lewis lung carcinoma cells, by American Type Culture Collection (2 mentions)
Cd11c microbeads, by Miltenyi Biotec (2 mentions)
Fetal bovine serum (fbs), by Biowest (2 mentions)
Penicillin streptomycin stock, by Lonza (2 mentions)
B16f10 cells, by American Type Culture Collection (1 mentions)
Recombinant tgf β1, by R&D Systems (1 mentions)
12 well transwell, by Corning (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Ll 2 llc1, by American Type Culture Collection (1 mentions)
Mle 12, by American Type Culture Collection (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Ccl 10, by American Type Culture Collection (1 mentions)
Raw264.7 cells tib 71, by American Type Culture Collection (1 mentions)
Crl 6322, by American Type Culture Collection (1 mentions)
Crl 3273, by American Type Culture Collection (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
32 protocols using crl 1642
1
Syngeneic Melanoma and Lung Carcinoma Treatment
2
Inhibition of Cancer Cell Migration
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B16F10 cells (CRL-6475, ATCC) and LLC cells (CRL-1642, ATCC) were cultured in Dulbecco’s modified Eagle’s medium high glucose (GIBCO, Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine serum (FBS). Cells were pre-treated with 10 μM AA or/and 100 μM NG before they were stimulated with 5 ng/mL recombinant TGF-β1 (R&D Systems, MN, USA).
Wound-healing assay was performed when cell density reached about 80% confluence. Scratches were made with pipette tips and then cultured for another 24 h. The width of the wound was measured at 0, 6, 12, and 24 h after making the scratches and analyzed with ImageJ software (National Institutes of Health).
To perform migration assay, we starved LLC cells for 24 h before seeding them with serum-free medium in the 12-well transwell with 8.0 μm pore size (Corning, NY, USA). The transwell inserts were placed in receiver wells with medium containing 10% FBS and cultured for another 24 h. Non-migrated cells left in the transwell were swabbed before fixation and Giemsa staining. Then, the transwell membrane was cut and mounted for counting the migrated cells.
250 LLC cells were seeded in a 6-well plate and treated with AA or/and NG for colony-formation assay. When visible colonies appeared, cells were fixed with methanol for 10 min, followed by Giemsa staining. The number of colonies were then counted.
Wound-healing assay was performed when cell density reached about 80% confluence. Scratches were made with pipette tips and then cultured for another 24 h. The width of the wound was measured at 0, 6, 12, and 24 h after making the scratches and analyzed with ImageJ software (National Institutes of Health).
To perform migration assay, we starved LLC cells for 24 h before seeding them with serum-free medium in the 12-well transwell with 8.0 μm pore size (Corning, NY, USA). The transwell inserts were placed in receiver wells with medium containing 10% FBS and cultured for another 24 h. Non-migrated cells left in the transwell were swabbed before fixation and Giemsa staining. Then, the transwell membrane was cut and mounted for counting the migrated cells.
250 LLC cells were seeded in a 6-well plate and treated with AA or/and NG for colony-formation assay. When visible colonies appeared, cells were fixed with methanol for 10 min, followed by Giemsa staining. The number of colonies were then counted.
3
Generation and Characterization of Murine Tumor Cell Lines
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BrafV600EPten−/−(BPD6, male) cell lines were generated and cultured in DMEM with 10% FBS at 37°C as previously described (Holtzhausen et al., 2015 (link)). KrasG12Dp53flox/flox cell lines were generated through enzymatic and mechanical digestion of autochthonous mouse lungs during week 16 post-virus administration by visual tumor dissection and serial culturing in DMEM with 10% FBS to remove fibroblasts. Cell lines were confirmed by rt-PCR and western blot. Lewis Lung Carcinoma cells were purchased from the ATCC (ATCC, CRL-1642) and cultured according to the supplier’s specifications. Bone marrow-derived dendritic cells (BMDCs) were harvested and differentiated using IL-4 (BioAbChem, 42-IL4 C) and GM-CSF (BioAbChem, 42-GMCSF) as previously described and purified using CD11c microbeads (Miltenyi Biotec, 130-108-338) according to the manufacturer’s protocol (Inaba et al., 1992 (link)). The HEK293-LEF1/TCF-luciferase cell line was cultured in DMEM with 10% FBS as previously described in the presence or absence of OMP-18R5 or OMP-54F28 (Ring et al., 2011 (link)). The murine dendritic cell line, DC2.4 (a generous gift from Dr. Kenneth Rock, University of Massachusetts Medical School) was cultured and generated as previously described (Shen et al., 1997 (link)).
4
Establishment of Murine Lung Cancer Model
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The murine Lewis lung carcinoma-1 (LLC-1) cell line was obtained from ATCC in 2018 (ATCC, catalog no. CRL-1642, RRID:CVCL_4358) and maintained in DMEM with 10% FBS and 1% Pen/Strep. LLC-1 cells were infected with lentivirus FUGW (29 (link)) to express firefly luciferase and GFP to obtain LLC-1 Luciferase expressing cells for bioluminescence imaging (BLI; LLC-1 Luc). LLC-1 cells bear a heterozygous KrasG12C mutation (25 (link)). All cell lines were regularly checked for mycoplasma contamination every other week using MycoAlert Mycoplasma Detection Kit (Lonza). Cell lines were cultured for a period of ∼ 2 months, after which they were replaced by new thaws. Cells were purchased from ATCC for this study specifically and not additionally authenticated by STR.
5
Generation and Characterization of Murine Tumor Cell Lines
6
Murine Lewis Lung Carcinoma Model
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All experiments were approved by the Danish Animal Experimental Inspectorate (Licence 2016‐15‐0201‐01043). The mice were housed with nesting material at ambient temperature (22 ± 2°C), 12:12‐h light–dark cycle. Standard rodent chow diet (Altromin No. 1324; Chr. Pedersen, Denmark) and water were provided ad libitum. Mice were single‐housed 5 days prior to cancer inoculation to avoid fighting between the mice during tumour progression and enabled food intake registrations. As described previously,23 Lewis lung carcinoma (LLC) cells (ATCC® CRL1642™) were cultured (5% CO2, 37°C) in Dulbecco's modified Eagle's medium (DMEM), high glucose (#41966‐029, Gibco, USA) with supplementation of 10% foetal bovine serum (FBS; #F0804, Sigma‐Aldrich, USA) and 1% penicillin–streptomycin (#15140122, ThermoFisher Scientific, USA). The cells were trypsinized and washed twice with sterile saline before the inoculation into the mice. The mice were subcutaneously inoculated at the flank with or without 2.5 * 105 LLC cells. All mice depicting ulcerations were sacrificed via cervical dislocation and excluded from the study.
7
Murine Lewis Lung Carcinoma Cell Culture
8
Characterization of Tumor Cell Lines
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Mouse Lewis lung carcinoma (LLC; ATCC, catalog no. CRL-1642, RRID:CVCL_4358) cell line was purchased from ATCC (RRID:SCR_001672). LLC-eGFP cell line was a gift from Professor Robert Brink. B16F10–3C melanoma cell line (13 (link)) was a gift from Professor Wolfgang Weninger. Murine AT-3 mammary carcinoma (RRID:CVCL VR89) cell line was a gift from Dr. Scott Abrams. KPC primary pancreatic ductal adenocarcinoma (PDAC) cell line (RRID:CVCL_XD11; ref. 14 (link)) was a gift from Professor Paul Timpson.
9
Heterotopic Lung Carcinoma Mouse Model
10
Murine Lung Cancer Cell Culture
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The Lewis lung carcinoma cells (LL/2) LL/2 cell line that is syngeneic for C57BL/6 mice (ATCC CRL-1642™) was purchased from ATCC as a model to study human lung cancer that is resistant to PD-1 blockade. The aforementioned tumor cell line was cultured at 37°C under 5% CO2 complete medium Dulbecco’s Modified Eagle Medium (DMEM), High Glucose and Glutamax, which is supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, 2 mM L-glutamine, 1 mM sodium pyruvate and non-essential amino acids (all reagents from Gibco). Bone marrow-derived macrophages (BMDMs) were isolated from femurs of wild-type mice of C57BL/6J background. Using a 26 G ½” needle, bone marrow cells were flushed out of the bones with Iscove’s modified Dulbecco’s medium (IMDM), supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% non-essential amino acid, 1% sodium pyruvate, 1% glutamine, and 20% (v/v) conditioned-media from L929 cells. Red blood cells were lysed using a 160 mM NH4CL and 170 mM Tris solution for 5 min at RT. Three to six × 106 viable cells were plated in non-cell-culture-treated petri dishes and grown for 5–7 d in the above mentioned fully supplemented IMDM medium.
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