When MCF-10A cell confluence reached about 80 %, single cells were obtained by 0.25 % typsin/EDTA (Gibco, USA) digestion, stained by MUC1-PE (BD Pharmingen, USA) and ESA-FITC (BD Pharmingen, USA). MUC1−ESA+ subpopulation was sorted by fluorescence-activated cell sorting (FACS, MoFlo, Dako-Cytomation, USA). The rest proportion of MCF-10A cells excluding MUC1−ESA+ was also sorted as control counterparts. Through FACS sorting, MUC1−ESA+ subpopulation was highly purified (purity greater than 98 %).
Typsin edta
Manufactured by Thermo Fisher Scientific
Sourced in United States
Trypsin-EDTA is a cell dissociation reagent used to detach adherent cells from cell culture surfaces. It contains the enzyme trypsin, which cleaves cell-cell and cell-substrate adhesion proteins, as well as EDTA, which chelates calcium and magnesium ions required for cell-cell adhesion. This product facilitates the harvesting of cells for subculturing, analysis, or other applications.
Automatically generated - may contain errors
Lab products found in correlation
Pen strep, by Thermo Fisher Scientific (4 mentions)
High glucose dmem, by Thermo Fisher Scientific (4 mentions)
Hek293t, by American Type Culture Collection (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Esa fitc, by BD (1 mentions)
Axiocam erc5s camera, by Zeiss (1 mentions)
Labscope software, by Zeiss (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
8 protocols using typsin edta
1
Isolation of MUC1-ESA+ Subpopulation
2
Mammalian Cell Culture Protocol
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All mammalian cells were cultured at 37°C with 5% CO2, and were maintained in high glucose DMEM (Gibco cat. no. 11965) for HEK293T (from ATCC) and NIH/3T3 (a gift from T. Reh’s lab at the University of Washington) cells, both supplemented with 10% FBS and 1× Pen/Strep (Gibco cat. no. 15140122; 100U/ml penicillin, 100 μg/ml streptomycin). Cells were trypsinized with 0.25% typsin-EDTA (Gibco cat. no. 25200–056) and split 1:10 three times a week.
3
Mammalian Cell Culture Protocol
4
Isolation and Culture of Tumor Cells
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Cells were maintained in DMEM + 10% fetal bovine serum (FBS, Axenia Biologix, Dixon, CA USA) and penicillin/streptomycin (Gibco, Waltham, MA USA) at 37°C under 10% CO2 and were were routinely passaged using 0.05% typsin-EDTA (Gibco). Primary NDL tumor cells were dissociated by enzymatic and mechanical methods essentially as described for normal mammary tissue42 (link) from Muc4wt/NDL (n=4) and Muc4ko/NDL (n=4) tumors. Tumor cells were harvested from the most advanced tumor and used within four passages to ensure equal comparison. The expression of Muc4 was retained over this duration, as confirmed by immunocytochemistry (data not shown).
5
Isolation and Culture of Tumor Cells
6
Transfection and Immunoblot of HEK293 Cells
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HEK293 cells (Stratagene; catalog no.: 240073) were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, nonessential amino acids (Gibco; catalog no.: 11140-035; 1:100 dilution), sodium pyruvate (Gibco; catalog no.: 11360-039; 1:100 dilution), and 50 U/ml penicillin–streptomycin, and passaged when they reached ∼60 to 70% confluency using typsin/EDTA (Gibco; catalog no.: 25300-054). For immunoblot analyses, cells were plated at a density of 62,600 cells/cm2 in a 12-well plate and cotransfected 24 h later with 1 μg pAAV-EGFP.T2A.NCLX and 1 μg pAAV-shCTRL, pAAV-shNCLX, pAAV-shNCLX-2, or none of these, or with 1 μg pAAV-mCherry.NLS or 1 μg pAAV-NCLX.Flag using 2 μl Lipofectamine 2000 Reagent (Invitrogen; catalog no.: 11668-019) following the manufacturer's instructions. Cells were imaged 24 h later using a Vert.A1 upright microscope outfitted with an Axiocam ERc 5s camera and LabScope software (all Zeiss) and then harvested for subsequent immunoblot analysis.
7
Routine Mammalian Cell Culture
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All mammalian cells were cultured at 37°C with 5% CO2, and were maintained in high glucose DMEM (Gibco cat. no. 11965) for HEK293T and NIH/3T3 cells or DMEM/F12 medium for A549 cells, both supplemented with 10% FBS and 1X Pen/Strep (Gibco cat. no. 15140122; 100U/ml penicillin, 100 μg/ml streptomycin). Cells were trypsinized with 0.25% typsin-EDTA (Gibco cat. no. 25200-056) and split 1:10 three times per week.
8
Routine Mammalian Cell Culture
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