Three different breast cancer cell lines were included in this study to represent a variety of phenotypes: MDA-MB-231 (triple-negative, but expected to express EGFR; ATCC® HTB-26™), MDA-MB-468 (triple-negative, but over-expresses EGFR; ATCC® HTB-132™), and AU565 (over-expresses HER2, and expected to express EGFR; ATCC® CRL-2351™). All cell lines were incubated for the duration of experiments in 37 °C and 5% CO2 level. Dulbecco’s modified Eagle’s medium low glucose (DMEM), containing L-glutamine, sodium pyruvate, and glucose was used for MDA-MB-231 and MDA-MB-468 cells. RPMI 1640 medium was used for AU565. All media were supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were sub-cultured after reaching > 80% confluency and were discarded after ~40 passages.
Htb 132
Manufactured by American Type Culture Collection
Sourced in United States
HTB-132 is a cell line obtained from human breast cancer tissue. It is a commonly used in vitro model for breast cancer research.
Automatically generated - may contain errors
Lab products found in correlation
Htb 26, by American Type Culture Collection (7 mentions)
Mda mb 468, by American Type Culture Collection (4 mentions)
Mda 231, by Thermo Fisher Scientific (3 mentions)
Mda 468, by Thermo Fisher Scientific (3 mentions)
Mycoalert, by Lonza (3 mentions)
Crl 2351, by American Type Culture Collection (2 mentions)
Hcc1806, by American Type Culture Collection (2 mentions)
Crl 2336, by American Type Culture Collection (2 mentions)
Hcc1937, by American Type Culture Collection (2 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Mda 157, by Thermo Fisher Scientific (2 mentions)
Dmem high glucose with glutamax, by Thermo Fisher Scientific (2 mentions)
Mda 468, by American Type Culture Collection (2 mentions)
Crl 2335, by American Type Culture Collection (2 mentions)
Htb 24, by American Type Culture Collection (2 mentions)
680 microplate reader, by Bio-Rad (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Crl 2315, by American Type Culture Collection (1 mentions)
Ccl 243, by American Type Culture Collection (1 mentions)
16 protocols using htb 132
1
Comparative Analysis of Breast Cancer Cell Lines
2
Cytotoxicity Assay of Peptide on Breast Cancer and Fibroblast Cells
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Cytotoxicity assays were performed as previously described in Rodriguez et al.22 (link) Briefly, breast cancer cell line MDA-MB-468 (ATCC® HTB-132™) cells or young fibroblast primary cells (100 μL; 1 × 104 cells per well) were seeded in 96-well flat-bottom tissue culture treated plates and were incubated at 37 °C in a 5% CO2 humidified atmosphere for 14 h, allowing for cell adhesion. Then the media was removed and 50 μL of supplemented RPMI medium (10%) and 50 μL of peptide (200, 100, 50, 25, 12.5 and 6.25 μg mL−1) were added, and the final FBS concentration was 5%. The plates were incubated for 2, 24, and 48 h at 37 °C in a 5% CO2 humidified atmosphere (physiologic pH), and cell viability was determined using the MTT assay. For this, 10 μL of MTT solution (5 mg mL−1) was added to each well, and the plates were incubated for 4 h at 37 °C. Formazan crystals were dissolved in DMSO (100 μL), and the absorbance (570 nm) was measured using a Bio-Rad 680 microplate reader (n = 3).
3
Cell Culture of TNBC and K562 Lineages
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TNBC cell lines HCC-1806 (ATCC, CRL 2335™), HCC-1937 (ATCC, CRL-2336™), HCC-70 (ATCC, CRL-2315™), and MDA-MB-468 (ATCC, HTB-132™), and the chronic myelogenous leukemia-derived K562 cell line (ATCC, CCL-243™) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cell lines were authenticated by DNA profiling using short tandem repeat (STR) analysis on an AmpFlSTR® Identifier™ PCR Amplification System at the National Institute of Genomic Medicine (INMEGEN), Mexico City, Mexico. The cells were used between passage 3 and passage 15. All the cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (Biowest SAS, Nuaillé, France) that was supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (Biowest SAS, Nuaillé, France), 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA). The cells were maintained in a 5% CO2 humidified atmosphere at 37 °C.
4
Myelinating Schwannoma and Breast Cancer Cell Cultures
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P0-expressing RT4 D6P2T myelinating Schwannoma cells (ATCC® CRL-2768™; [35 (link)]) and non-P0-expressing MDAMB 468 human breast cancer cells (ATCC® HTB-132™) were grown in Dulbecco’s modified Eagle medium (Life Technologies, UK) containing penicillin, streptomycin and foetal calf serum (All BD Biosciences) at 37 °C and 5% CO2.
In line with their use by Whitney et al. [27 (link)], transgenic B6.Cg-Tg(Thy1-YFP)-16Jrs/J (THY-1 YFP) mice were obtained from JAX (the Jackson Laboratory) and were used for ex vivo and in vivo nerve staining and in vivo biodistribution studies (8–15 weeks old). THY-1 YFP mice express spectral variants of GFP (yellow fluorescent protein—YFP; ex 488, em 520) at high levels in motor and sensory neurons. The fluorescent signal in the nerves was used as an internal control for the staining (pattern) of the developed imaging agents. Balb/c nude mice were used as non-fluorescent control.
In line with their use by Whitney et al. [27 (link)], transgenic B6.Cg-Tg(Thy1-YFP)-16Jrs/J (THY-1 YFP) mice were obtained from JAX (the Jackson Laboratory) and were used for ex vivo and in vivo nerve staining and in vivo biodistribution studies (8–15 weeks old). THY-1 YFP mice express spectral variants of GFP (yellow fluorescent protein—YFP; ex 488, em 520) at high levels in motor and sensory neurons. The fluorescent signal in the nerves was used as an internal control for the staining (pattern) of the developed imaging agents. Balb/c nude mice were used as non-fluorescent control.
5
PRMT Inhibitor Screening in Cancer Cell Lines
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MDA-MB-468 breast carcinoma cells (ATCC; HTB132), HCT116 +/+ p53 colon carcinoma cells were grown in DMEM supplemented in 5% FBS. HepaRG terminally differentiated liver cell line (ThermoScientific; HPRGC10) and HepaRG-Fucci cells were grown in Williams media supplemented with Glutamax and 5% FBS and normal HMECs (ATCC; PCS-600-010) were grown in mammary epithelial basal medium (ATCC; PCS-600-030) supplemented with mammary epithelial cell growth kit (ATCC; PCS-600-040). Cells were recovered with 0.25% trypsin-EDTA, plated on cell culture treated plates, and/or coverslips according to the experimental design and incubated overnight in 37 °C humidified CO2 incubator kept at 5% CO2 (800WJ; ThermoScientific). Cells were washed twice with sterile PBS before switching to serum-free treatment media along with varying concentrations of the PRMT inhibitor compound (0–40 μM).
6
Quantifying Wnt3A Protein Activation
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MDA-468 cells (ATCC® HTB-132™) were stably transfected with a luciferase reporter of the canonical Wnt signaling (Addgene, #24308). MDA-468-luciferase cells were plated in 96 well plate (5000 cells/well) in DMEM medium plus 10% FBS for 24 hours. Then, 150 μL fresh DMEM plus 10% FBS and 50 μL L-Wnt-3a-cell-conditioned medium were added and incubated for another 18 hours. The medium was then removed, and cells were washed with PBS once before 200 μL cell lysis buffer was added and incubated for 10 minutes at room temperature. 50 μL cell lysates, 50 μL luciferase substrate A and 50 μL luciferase substrate B from the luciferase assay kit were mixed and the light signals were immediately read with a luminometer. The light intensity was calibrated to a standard curve to calculate the amount of Wnt3A proteins.
7
Culturing and Starvation of Cancer Cell Lines
8
Tumor-Targeted Superantigen Treatment of Breast Cancer
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Human breast carcinoma cell MDA-MB-468 (obtained from the ATCC: ATCC® HTB-132™) were seeded in a 96-well plate (6.4 × 103 cell/well) and incubated at 37 °C in 5% CO2 overnight to allow cell attachment to the well. Cells were treated with 800 ug/mL of endotoxin-free SPEA superantigen, 200 ug/mL SPEA-based peptides, or tumor-targeted superantigen-based peptides. Then, 3.2 × 104 PBMCs were added to wells with tumor cells and SAg derivatives, and incubated for 48 h at 37 °C incubator with 5% CO2. The cell lysate was removed and used for cytokine assessment. Cells were fixed with 3.8% Formaldehyde solution for 10 min and then stained with DAPI. The number of viable tumor cells was measured by ArrayScan™ XTI (Thermo Fisher Scientific, Waltham, MA, USA) where an automated quantitation of DAPI positive nuclei was performed using the target activation module, and the apoptotic nuclei were distinguished and excluded by their collapsed size, higher chromatin intensities, and nuclear fragmentation [84 (link)]. The PBMCs were also excluded by size. Cell count is presented as a percentage of tumor + PBMCs (TP) will count.
9
MDA-MB-231 and MDA-MB-468 Cell Culture
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Breast cancer cell line MDA-MB-231 and MDA-MB-468 was obtained from the ATCC® (reference HTB-26™ and HTB-132™; Gaithersburg, MD). The cell lines were cultivated in DMEM high glucose medium (Gibco; Gaithersburg, MD) with a final concentration of 10% fetal bovine serum (FBS) (Gibco) and 1% antibiotic–antimycotic (Gibco). Cells were trypsinized every 3 to 4 days, never exceeding 10 passages in all experiments, and maintained at 37 °C and 5% CO2.
10
Characterization of Breast Cancer Cell Lines
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A panel of human breast cancer cell lines were selected for this study to represent a variety of characteristics and receptor expression profiles: (i) MDA-MB-231 (MDA231; triple negative, but expected to express EGFR; ATCC ® HTB-26™); (ii) MDA-MB-231, KRas-CRM (MDA231K; same as MDA-MB-231, certified for KRas mutation; ATCC ® CRM-HTB-26™);
(iii) MDA-MB-468 (MDA468; triple negative, but over-expresses EGFR; ATCC ® HTB-132™);
(iv) AU565 (over-expresses HER2, and expected to express EGFR; ATCC ® CRL-2351™); (v) MCF7 (estrogen and progesterone-positive, and expected to express HER2 and EGFR; ATCC ® HTB-22™), and; (vi) MDA-MB-435 (MDA-435): Long known as an estrogen-independent breast cancer cell line expressing HER-2 [25, 26] . MDA-435 cells have been source of controversy since it bears both epithelial and melanocytic markers [27, 28] . All cell lines except AU565 were cultured in DMEM low glucose medium, while AU565 was cultured in RPMI-1640 (both mediums supplemented with 10% (v/v) FBS, 100 U/mL penicillin and 100 μg/ml streptomycin). All cell lines were maintained at 37C and 5% CO 2 and were sub-cultured after reaching 80-90% confluency. Cells were discarded after 40 passages, and frozen cells (stored in liquid nitrogen) were thawed as replacement.
(iii) MDA-MB-468 (MDA468; triple negative, but over-expresses EGFR; ATCC ® HTB-132™);
(iv) AU565 (over-expresses HER2, and expected to express EGFR; ATCC ® CRL-2351™); (v) MCF7 (estrogen and progesterone-positive, and expected to express HER2 and EGFR; ATCC ® HTB-22™), and; (vi) MDA-MB-435 (MDA-435): Long known as an estrogen-independent breast cancer cell line expressing HER-2 [25, 26] . MDA-435 cells have been source of controversy since it bears both epithelial and melanocytic markers [27, 28] . All cell lines except AU565 were cultured in DMEM low glucose medium, while AU565 was cultured in RPMI-1640 (both mediums supplemented with 10% (v/v) FBS, 100 U/mL penicillin and 100 μg/ml streptomycin). All cell lines were maintained at 37C and 5% CO 2 and were sub-cultured after reaching 80-90% confluency. Cells were discarded after 40 passages, and frozen cells (stored in liquid nitrogen) were thawed as replacement.
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