All mouse experiments were performed in accordance with the protocol of Institutional Animal Care and Use Committee, KAIST. Xenograft models were prepared by subcutaneously injecting 1×106 A375 human melanoma cells (ATCC CRL-1619) into the shaved right flank of 8–12 weeks old male NSG mice (Jackson Laboratory). After implantation, the mice were randomly allocated to different experimental groups and 2.5×106 T cells that were purified based on dLNGFR expression (equivalent to 1×106 1G4+ TCR-T cells) were intravenously injected into each mouse on day 7. Tumor length and width were measured using a digital caliper, and tumor volume (V) was calculated as V=1/6 × π×length × width × (length+width)/2. Mice were euthanized when the major axis of the tumor reached ~20 mm. The tumor burden of each mouse was measured at 7-day intervals using a bioluminescent in vivo imaging system (IVIS) and quantified as the luminescence of the region of interest (generally the whole area of one mouse).
A375 human melanoma cells
Manufactured by American Type Culture Collection
Sourced in United States
A375 human melanoma cells are a well-characterized cell line derived from a human malignant melanoma. They are commonly used for research purposes in the study of melanoma biology and the development of anti-cancer treatments.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Nsg mice, by Jackson ImmunoResearch (1 mentions)
Bi 6727, by Chemietek (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Mrc 5 human lung fibroblasts, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
A549 cell, by American Type Culture Collection (1 mentions)
Rpmi 1640 medium, by Corning (1 mentions)
Melanocyte growth supplement, by ScienCell (1 mentions)
Raw 264.7 murine macrophage, by American Type Culture Collection (1 mentions)
B16f10 murine melanoma cells, by American Type Culture Collection (1 mentions)
In vitro transcription kit, by Roche (1 mentions)
O dianisidine, by Merck Group (1 mentions)
Anti dig antibody, by Roche (1 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
12 protocols using a375 human melanoma cells
1
Melanoma Xenograft Tumor Model in NSG Mice
2
Culturing A375 Human Melanoma Cells
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A375 human melanoma cells (ATCC, VA) were
cultured in HyClone Dulbecco’s modified Eagle’s medium
(DMEM, Thermo, CA) supplemented with 10% fetal bovine serum (FBS,
Thermo, CA). Cells were maintained in a humidified incubator with
5% CO2 at 37 °C.
cultured in HyClone Dulbecco’s modified Eagle’s medium
(DMEM, Thermo, CA) supplemented with 10% fetal bovine serum (FBS,
Thermo, CA). Cells were maintained in a humidified incubator with
5% CO2 at 37 °C.
3
BI 6727 Treatment on A375 Melanoma Cells
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1 × 106 A375 human
melanoma cells (ATCC, VA) were seeded in 100 mm tissue culture plates
(TPP, MO) containing 10 mL of supplemented DMEM (Thermo, CA) and were
allowed to recover in untreated medium for 24 h under standard cell
culture conditions. Following recovery, medium was aspirated, and
cells were treated with 25 nM BI 6727 (Chemietek, IN) or vehicle control
(DMSO) in supplemented DMEM. After 24 h, cells were collected by trypsin
digestion and centrifugation at 300g for 3 min at
4 °C. Supernatant was removed and cell pellets were washed three
times with PBS. The treatments were performed using identical procedures
on two varying A375 cell passages three times each for a total of
six experimental replicates per treatment group. Cell pellets were
stored at −80 °C prior to cell lysate preparation.
melanoma cells (ATCC, VA) were seeded in 100 mm tissue culture plates
(TPP, MO) containing 10 mL of supplemented DMEM (Thermo, CA) and were
allowed to recover in untreated medium for 24 h under standard cell
culture conditions. Following recovery, medium was aspirated, and
cells were treated with 25 nM BI 6727 (Chemietek, IN) or vehicle control
(DMSO) in supplemented DMEM. After 24 h, cells were collected by trypsin
digestion and centrifugation at 300g for 3 min at
4 °C. Supernatant was removed and cell pellets were washed three
times with PBS. The treatments were performed using identical procedures
on two varying A375 cell passages three times each for a total of
six experimental replicates per treatment group. Cell pellets were
stored at −80 °C prior to cell lysate preparation.
4
Culturing Common Cell Lines
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A549 cells (non-small cell lung cancer; CCL-185) were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium (Corning) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco). A375 human melanoma cells (CRL-1619) were also obtained from ATCC and cultured in high-glucose DMEM (Gibco) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco). MRC-5 human lung fibroblasts (CCL-171) and BJ foreskin fibroblasts (CRL-2522) were also obtained from ATCC and maintained in EMEM (ATCC) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco). HEK293T cells were obtained from GenHunter and cultured in high-glucose DMEM (Gibco) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin–streptomycin (Gibco). All cell lines were maintained at 37 °C and 5% CO2. All cell lines were routinely tested for mycoplasma and were at all times mycoplasma negative.
5
Culturing Diverse Cell Lines for Research
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B16F10 murine melanoma cells, 4T1 murine mammary carcinoma cells, HUVEC human endothelial cells, RAW264.7 murine macrophages, YUMM1.7 murine melanoma cells, A375 human melanoma cells, C32 human melanoma cells and murine Lewis Lung Carcinoma (LLC) cells designated LL/2 (LLC1) were obtained from the American Type Culture Collection (ATCC; cat. no. CRL-1642). Human Dermal Fibroblast (HDF) were obtained from ScienCell Research Laboratories, Inc. and cultured in 2% FBS in growth medium supplemented with Fibroblast Growth Supplements from ScienCell Research Laboratories, Inc. YUMM1.7 cells were cultured in DMEM F-12 medium in presence of 10% FBS, 1% NEAA and 1% Pen-Strep. 4T1 cells were cultured in RPMI-1640 medium in the presence of 5% FBS, 1% Pen-Strep and 1% sodium pyruvate. HUVECs were cultured in VCBM media plus Endothelial Cell Growth Kit. Β16F10, RAW264.7, C32, A375 and LLC cells were cultured in DMEM plus 10% FBS 1.0% Pen-Strep and 1.0% sodium pyruvate. Human primary melanocytes were obtained from ScienCell Research Laboratories, Inc. and cultured in Melanocyte Media containing 0.5% FBS and Melanocyte Growth Supplement.
6
Investigating Cellular Effects of 5-CHO-THF
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5-formyltetrahydrofolate (5-CHO-THF) was purchased from Schircks Laboratories (Bauma, Switzerland). Fetal bovine serum (FBS) and trypsin-EDTA were purchased from Invitrogen, Thermo Fisher Scientific Inc. (CA, USA). dNTP was purchased from FocusBio (CA, USA). The in vitro transcription kit, anti-DIG antibody, and NBT-BCIP used for WISH were purchased from Roche (Basel, Switzerland). The A375 human melanoma cells and A549 alveolar basal epithelial adenocarcinomic cells, originally from the American Type Culture Collection (ATCC), were purchased from the Bioresource Collection and Research Center (BCRC). Phospho-Histone H3 (pH3) antibody was from Santa Cruz Biotechnology Inc. (SC-374669, DA, USA). Goat anti-mouse IgG Alexa Flour® 488 was from Abcam plc. (ab150113, Cambridge, UK). All other chemicals, including N-acetyl-L-cysteine (NAC), U0126, folic acid (FA), N-phenylthiourea and o-dianisidine were obtained from Sigma-Aldrich Chemical Co. (WI, USA). The HPLC gel filtration column AQUASIL C18 was from Thermo Fisher Scientific Inc. (MA, USA).
7
Generating Fluorescent Melanoma Cells
8
Culturing Human Cancer Cell Lines
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The human tumor cell lines (A375 human melanoma cells, Cat no. ATCC® CRL-1619™; A549 human lung carcinoma cells, Cat no. ATCC® CCL-185™; and MDA-MB-231 human breast carcinoma cells, Cat no. ATCC® HTB-26™) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) via LGC Standards GmbH, Wesel Germany. The B164A5 murine melanoma cell line and cell culture media, and specific supplements were purchased from Sigma-Aldrich Co. (Ayrshire, UK). Human keratinocytes (HaCaT) were a gift from the University of Debrecen, Debrecen, Hungary. The cells were cultured in Dulbecco's modified Eagle's Medium (DMEM) containing 4.5 g/l glucose, L-glutamine and NaHCO3, and supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal bovine serum (FBS). Cells were kept under standard conditions (humidified atmosphere, 5% CO2, 37°C) and passaged every second day. Cell number was determined by using the cell counting chamber (Neubauer) in the presence of Trypan blue.
9
Melanoma Cell Tyrosinase Extraction Protocol
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A375 human melanoma cells were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). A375 cells were grown in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Burlington, ON, Canada) supplemented with L-glutamine, 10% (v/v) fetal bovine serum (Invitrogen), 50 µg/mL streptomycin (Sigma-Aldrich), 50 units/mL penicillin (Sigma-Aldrich) and supplemented with 200 µM of L-tyrosine for tyrosinase induction. Cell cultures were incubated at 37°C, in a humidified atmosphere of 5% CO2. Cells were scraped out from the tissue culture plate with phosphate-buffered saline (PBS) and were homogenized at 4°C in PBS. The homogenate was centrifuged at 1,000× g for 10 min. The precipitate was sonicated in PBS on ice and the mixture was centrifuged at 10,000× g for 30 min. The supernatant containing tyrosinase was used for the measurement of the inhibitory effects.
10
Melanoma Cell Culture and Tyrosinase Extraction
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A375 human melanoma cells were obtained from American Type Culture Collection (ATCC; Rockville, MD, USA). A375 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Burlington, ON, Canada) supplemented with L-glutamine, 10% (v/v) fetal bovine serum (FBS; Invitrogen), 50 µg/mL streptomycin (Sigma Aldrich, St. Louis, MO, USA), 50 units/mL penicillin (Sigma Aldrich) and supplemented with 200 µM of l -tyrosine for tyrosinase induction. Cell cultures were incubated at 37 °C, in a humidified atmosphere of 5% CO2. Cells were scraped out from the tissue culture plate with phosphate-buffer saline (PBS) and were homogenized at 4 °C in PBS. The homogenate was centrifuged at 1000× g for 10 min. The precipitate was sonicated in PBS on ice and the mixture was centrifuged at 10,000× g for 30 min. The supernatant containing tyrosinase was used for the measurement of the inhibitory effects.
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