Patient-derived colon cancer cell lines (CRC1, CPP-14/25/43/6/19/30/36) were derived from CRC surgeries provided by CHU-Carémeau (Nîmes, France, ClinicalTrial.gov Identifier#NCT01577511) within an approved protocol by the french Ethics Committee: CPP (Comité de Protection des Personnes) Sud Méditerrannée III. We have complied with all relevant ethical regulations for work with human participants, and informed written consent was obtained for all the patients. CRC1, CPP-14/25/43 cell lines were derived from primary tumors and CPP-6/19/30/36 from metastatic tumors. CTC44 and CTC45 are circulating tumor cell lines derived from blood of metastatic chemotherapy-naïve stage IV CRC patients31 (link). HCT-116 (ATCC® CCL-247™) and SW620 (ATCC® CCL-227™) are commercially available colon cancer cell lines derived, respectively from primary and metastatic human tumors.
Atcc ccl 247
Manufactured by American Type Culture Collection
Sourced in United States
ATCC CCL-247 is a cell line derived from a human colorectal adenocarcinoma. It is a commonly used model in cancer research.
Automatically generated - may contain errors
Lab products found in correlation
Hct116, by American Type Culture Collection (7 mentions)
Atcc htb 38, by American Type Culture Collection (5 mentions)
Atcc htb 22, by American Type Culture Collection (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ht 29, by American Type Culture Collection (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Rna isolation kit, by Qiagen (1 mentions)
Mtt reagent, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Cell cycle kit, by Thermo Fisher Scientific (1 mentions)
Ldh kit, by Promega (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Multiskan fc, by Thermo Fisher Scientific (1 mentions)
Hct116, by OriGene (1 mentions)
Rg217050, by OriGene (1 mentions)
Pcmv6 ac gfp tagged cloning vector, by OriGene (1 mentions)
Crl 1541, by American Type Culture Collection (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
27 protocols using atcc ccl 247
1
Characterization of Patient-Derived CRC Cell Lines
2
Colorectal Cancer Cell Line Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells of the human colorectal cell line HCT-116 (ATCC® CCL-247™) were purchased from the ATCC (Manassas, VA, USA). All tissue culture reagents, including fetal bovine serum and standard culture medium RPMI-1640, were purchased from Biological Industries (Beit Haemek, Israel). An LDH kit was purchased from Promega (Madison, WI, USA); a cell cycle kit was purchased from Thermo Fisher (Waltham, MA, USA); and an RNA isolation kit was purchased from QIAGEN (Hilden, Germany). MTT reagent and all other materials were purchased from Sigma Aldrich (St. Louis, MO, USA). HTE (aerial parts) was purchased from Al Alim—Medicinal Herb Center, Zippori, Israel.
3
Cytotoxicity Assay of Novel Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cytotoxicity of compounds 1 , 3 , 5 and 6 was measured through the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay33 (link), that is based in the conversion of the tetrazolium salt to a formazan product by viable cells. Two cell lines obtained from American Type Culture Collection (Manassas, Virginia, EUA) were used: HCT-116 (colorectal carcinoma, ATCC® CCL-247™) and MCF-7 (breast adenocarcinoma, ATCC® HTB-22). The assay was conducted essentially as described by Monteiro et al.34 (link). The HCT-116 and MCF-7 cells (1 × 104 cells well−1) were seeded in 96-well plates and, after 24 h, different concentrations (5 µM and 50 µM) of the compounds were added and incubated for 72 h. Doxorubicin (0.003 to 10 µM) and DMSO (0.5%) were used as a positive and negative controls, respectively. At the end of incubation, the supernatant was replaced with fresh medium containing MTT (0.5 mg/mL) for 3 h. The supernatant was, then, removed, and the MTT formazan product was dissolved in 150 μL DMSO. The absorbance was measured using a multiplate reader (Multiskan™ FC, Thermo Scientific, Finland) at 570 nm. All experiments were performed in duplicate. IC50 values, along with 95% confidence intervals, were calculated by non-linear regression using GraphPad Prism 5.0 (Intuitive Software for Science, La Jolla, CA, USA).
4
Establishment of DCLK1-overexpressing Colorectal Cancer Cells
5
Culturing HCT116 and CCD112 Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human HCT116 colorectal cancer (CRC) (ATCC CCL-247) and CCD112 normal colon (ATCC CRL-1541) cell lines were purchased from ATCC (VA, USA) and cultured according to ATCC’s recommendation [35 (link),36 (link)]. The cell lines were maintained in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco) under standard cell culture conditions.
6
Cytotoxicity Assessment of Mercury in HCT-116 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cytotoxicity of mercury was measured using HCT-116 cells (ATCC® CCL-247™, ATCC, Manassas, VA, USA) as a cell model of the intestine. HCT-116 cells were
cultured in McCoy’s 5A culture medium (ATCC) with 10% fetal bovine serum (FBS, Gibco, Burlington, ON, Canada) and penicillin-streptomycin (Gibco) at 37°C under 5% CO2conditions. For measurement of cytotoxicity, 1 mL of 5 × 105 HCT-116 cells were added to wells of a 12-well plate (Tissue Culture Test Plate 12, TPP, Trasadingen,
Switzerland) and incubated at 37°C under 5% CO2 for 24 hr. Cells were washed twice with PBS (pH 7.4), and 1 mL of culture media with Hg(II) solution was added to the
wells at final concentrations of 1, 2.5, 5, 7.5, 10, 50, and 100 µg/mL, respectively, before culturing at 37°C with 5% CO2 for 24 hr. As a control, 0.5 mM nitric acid
solution was used. The survival rate was expressed as a percentage of the number of viable cells in 0.5 mM of nitric acid solution (set to 100%). After treatment, cells were washed
twice with PBS, and the number of viable cells was counted by trypan blue staining.
cultured in McCoy’s 5A culture medium (ATCC) with 10% fetal bovine serum (FBS, Gibco, Burlington, ON, Canada) and penicillin-streptomycin (Gibco) at 37°C under 5% CO2conditions. For measurement of cytotoxicity, 1 mL of 5 × 105 HCT-116 cells were added to wells of a 12-well plate (Tissue Culture Test Plate 12, TPP, Trasadingen,
Switzerland) and incubated at 37°C under 5% CO2 for 24 hr. Cells were washed twice with PBS (pH 7.4), and 1 mL of culture media with Hg(II) solution was added to the
wells at final concentrations of 1, 2.5, 5, 7.5, 10, 50, and 100 µg/mL, respectively, before culturing at 37°C with 5% CO2 for 24 hr. As a control, 0.5 mM nitric acid
solution was used. The survival rate was expressed as a percentage of the number of viable cells in 0.5 mM of nitric acid solution (set to 100%). After treatment, cells were washed
twice with PBS, and the number of viable cells was counted by trypan blue staining.
7
HCT116 Cell Line Cultivation and Transfection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HCT116 cell line was purchased from the American Tissue Culture Collection (ATCC,
cat. no. ATCC CCL-247) and the hRpn13-deletion (ΔhRpn13) HCT116 cell line
generated in this study, as described below. Cells were grown in McCoy’s
5A modified medium (ATCC), supplemented with 10% fetal bovine serum
(Atlanta Biologicals) in a 37 °C humidified atmosphere of 5%
CO2. Plasmids were transfected using Lipofectamine LTX according
to the manufacturer’s instructions (Thermo Fisher Scientific).
cat. no. ATCC CCL-247) and the hRpn13-deletion (ΔhRpn13) HCT116 cell line
generated in this study, as described below. Cells were grown in McCoy’s
5A modified medium (ATCC), supplemented with 10% fetal bovine serum
(Atlanta Biologicals) in a 37 °C humidified atmosphere of 5%
CO2. Plasmids were transfected using Lipofectamine LTX according
to the manufacturer’s instructions (Thermo Fisher Scientific).
8
Establishing Colorectal Cancer Spheroids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human colorectal cancer cell lines HCT-116 and HT-29 were selected because of their reported sensitivity towards Artesunate [61 (link),62 (link)] and their spheroid formation ability [63 (link),64 (link),65 (link)]. Cell lines were purchased from ATCC (HCT-116, ATCC CCL-247; HT-29, ATCC HTB-38) and cultured according to the manufacturer’s instructions. Cell line-derived cancer spheroids were prepared as described previously [63 (link)]. Briefly, monolayer cultures with a confluency of at least 90% were detached with 0.05% trypsin/0.53 mM EDTA (Pan Biotech, Aidenbach, Germany). Cell number and viability were determined using the trypan blue exclusion assay. Cell suspensions with a viability of at least 90% were used for spheroid formation. 5 × 104 vital cells were seeded in each well of a low adhesive 96-well microtiter plate and incubated for 48 h under standard culture conditions (37 °C, 5% CO2). A single cancer spheroid was obtained in each well. Cancer cell lines were used for a maximum of 10 passages. Mycoplasma assays were performed quarterly.
9
Hypoxic Pretreatment of HCT116 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HCT116 cells were purchased from ATCC (ATCC CCL-247) and grown in McCoys 5A media supplemented with 10% FBS and 1% Penicillin and Streptomycin. Cells were grown in a normoxia incubator at 37 °C with 5% CO2. Cells were passaged with gentle trypsinization (0.25% trypsin with EDTA) and re-seeding at 1:10 ratio. Cells used in the experiments did not exceed passage 12. Cells were re-seeded in 12-well plates or T25 flasks for experiments. For epigenomic and transcriptomic experiments, cells were first seeded at a 1:12 ratio in T-25 flasks and allowed to adhere for 12 h in normoxia. After adherence, one set of cells continued to grow in normoxic conditions and another set was transferred to a hypoxic incubator for pre-treatment.
10
Cathepsin B Inhibition in Colon Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3-bromopyruvate (purity ≥97%) was purchased from Sigma-Aldrich (St. Louis, USA). Human liver CTSB (purity ≥95% by SDS-PAGE) was obtained from Calbiochem (Sigma-Aldrich). The specific fluorogenic substrate for CTSB, Z-Arg-Arg-AMC hydrochloride salt, was supplied by Bachem AG (Bubendorf, Switzerland). Human colorectal adenocarcinoma Caco-2 cells (ATCC ® HTB-37 ™ ) and human colorectal carcinoma HCT 116 cells (ATCC ® CCL-247 ™ ) were purchased from ATCC (Manassas, USA). Primary monoclonal IgG rat anti-human CTSB antibodies were purchased from R&D Systems (Minneapolis, USA). Secondary IgG (H+L) goat anti rat antibodies conjugated with Alexa Fluor 633 dye were purchased from Invitrogen (Carlsbad, USA). All other reagents were of analytical, LC/MS or other grade suitable for cell culturing.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!