Human breast cancer cells, MCF-7, were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). MCF-7 cells were grown in Dulbecco’s Modified Essential Medium (DMEM; Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Hyclone), 100 U/ml penicillin, and 100 μg/ml streptomycin (Hyclone). Cells were maintained at 37 °C in a humidified incubator with 5% CO2. To establish primary mammospheres, MCF-7 cells were seeded at a density of 3.5–4 × 104 cells/well in ultra-low attachment 6-well plates containing 2 ml of complete MammoCult™ medium (StemCell Technologies, Vancouver, BC, Canada) which was supplemented with 4 μg/ml heparin, 0.48 μg/ml hydrocortisone, 100 U/ml penicillin, and 100 μg/ml streptomycin. To establish secondary mammosphere, primary mammosphere was collected into individual conical tubes by gentle centrifugation at 1000 rpm for 1 min. After removal of supernatant, cell pellet was trypsinized with 1X EDTA/Trypsin, followed by replating a single cell suspension at a density of 4 × 104 cells/well in ultra-low attachment 6-well plates containing 2 ml of complete MammoCult™ medium [7 (link)]. In 5 days, the number and size of the mammosphere were assessed compared with control. The same procedure was repeated for tertiary mammospheres [7 (link)].
Streptomycin
Manufactured by STEMCELL
Sourced in Canada, United States
Streptomycin is a broad-spectrum antibiotic that is effective against a variety of bacterial species, including Gram-positive and Gram-negative bacteria. It is commonly used in research and laboratory settings to prevent bacterial contamination of cell cultures and other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by STEMCELL (12 mentions)
Penicillin, by Thermo Fisher Scientific (10 mentions)
Streptomycin, by Thermo Fisher Scientific (9 mentions)
Heparin, by STEMCELL (3 mentions)
Streptomycin, by American Type Culture Collection (3 mentions)
Iscove s modified dulbecco s media, by Thermo Fisher Scientific (2 mentions)
Stemspan serum free expansion medium 2, by STEMCELL (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by American Type Culture Collection (2 mentions)
C1498, by American Type Culture Collection (2 mentions)
Hydrocortisone, by STEMCELL (2 mentions)
Glutamine, by STEMCELL (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
M2 10b4 and s1 s1 stromal cells, by STEMCELL (2 mentions)
Fetal bovine serum (fbs), by STEMCELL (2 mentions)
Complete mammocult medium, by STEMCELL (1 mentions)
Mcf 7, by STEMCELL (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
G csf, by Thermo Fisher Scientific (1 mentions)
Stemspan serum free expansion medium 2 sfem 2, by STEMCELL (1 mentions)
13 protocols using streptomycin
1
Establishment of Mammosphere Cultures from MCF-7 Cells
2
Expansion and Electroporation of CD34+ Cells
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CD34 + cells were isolated from mononuclear cells derived from mobilized peripheral blood stem cells (PBSC) autologous transplant products by using EasySep Human CD34 Positive Selection Kit II (StemCell Technologies, 18056). Cell were cultured for 48 h before electroporation in StemSpan™ Serum-Free Expansion Medium II (SFEM II) (StemCell Technologies, 09605) with streptomycin (20 mg/mL), penicillin (20 unit/mL) and the following human cytokines (all from GenScript unless stated otherwise, catalog numbers and dilution used in parentheses): FLT3L (Z02926, 100 ng/mL), G-CSF (Z02980, 10 ng/mL), SCF (Z02692, 100 ng/mL), TPO (Pepro-Tech, 300-18, 25 ng/mL) and IL-6 (Z03034, 10 ng/mL). Cells were cultured at a density of 2.5*10^5 cells/ml in 96-well U-bottom plates.
3
Culturing Human and Mouse Immune Cells
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Human LSCs were cultured in StemSpan™ Serum-Free Expansion Medium II (STEMCELL, USA) with penicillin (100 U/mL), streptomycin (100 mg/mL), stem cell factor (SCF, 20 ng/ml), thrombopoietin (TPO, 20 ng/ml), Flt3-L (20 ng/ml), IL-3 (10 ng/ml), and IL-6 (10 ng/ml). Mouse LSCs were cultured in Iscove’s Modified Dulbecco’s Media (Thermo Fisher Scientific, USA) with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 mg/mL), SCF (20 ng/ml), TPO (20 ng/ml), Flt3-L (20 ng/ml), IL-3 (10 ng/ml), and IL-6 (10 ng/ml). Human and mouse ILC1s or NK cells were cultured in RPMI 1640 (Thermo Fisher Scientific, USA) with 10% FBS, penicillin (100 U/mL), streptomycin (100 mg/mL), IL-12 (10 ng/ml), and IL-15 (100 ng/ml). The mouse AML cell line C1498 (American Type Culture Collection) was cultured in RPMI 1640 with 10% FBS, penicillin (100 U/mL), and streptomycin (100 mg/mL). Cultures were incubated at 37°C in a humidified atmosphere of 5% CO2. All cytokines were from PeproTech. penicillin and streptomycin were from Thermo Fisher Scientific.
4
Cell Line Establishment and Authentication
5
Tumorsphere Formation and Paclitaxel Response
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For tumorsphere formation, cells were grown in MammoCultTM Human Medium supplemented with MammoCultTM Proliferation Supplement (StemCell Technologies, Vancouver, BC, Canada), 50 U/mL penicillin, 50 mM streptomycin, 4 µg/mL heparin (StemCell Technologies, Vancouver, BC, Canada) and 0.48 µg/mL hydrocortisone (StemCell Technologies, Vancouver, BC, Canada). Cells were plated in 24-well ultra-low attachment plates (Corning, Corning, NY, USA) at a density of 6000–20000 cells per well, depending on the cell line, and cultured for 5 days. To test the effect of paclitaxel, tumorspheres were treated with DMSO or 0.1 µM paclitaxel and they were collected after 48 h. The number of tumorspheres was quantified using an inverted microscope (Olympus, Tokyo, Japan).
6
Evaluating Metallic and ZnO Nanoparticle Toxicity on THP-1 Cells
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The human monocyte THP-1 cell line (ATCC TIB-202) was used to evaluate the toxicity levels of metallic- and ZnO-NPs as well as the combination of both NPs. THP-1 cells were grown as monocytes in RPMI medium supplemented with heat deactivated 10% fetal calf serum (Hyclone, GE, IL, USA), and a mixture of penicillin (100 μg/mL) and streptomycin (100 U/mL) (StemCell, Vancouver, BC, Canada). THP-1 cells were incubated at 37°C in a humid atmosphere of 5% CO2. Cells (1x104 cells/well) were activated the night before of the experiment by the addition of 40 ng/mL phorbol myristate acetate (PMA, Sigma). The next day, the medium was aspirated and replaced with fresh media without antibiotics and exposed to the NPs for 24 h using the same incubation temperature as stated above. Thereafter, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, Promega, WI, USA) was added to the wells and the optical density of the plates was read at 450 nm using a plate reader (Epoch, Tecan, Männedorf, Switzerland). Staurosporine (1 μg/mL, Sigma) and untreated cells were used as positive and negative controls, respectively. The cytotoxic activities were performed without exposure to the light to mimic the environment once the NPs are penetrating into the body.
7
Mammosphere Formation Assay
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Single cells were plated on ultralow attachment 6 well tissue culture plates (Costar, Catalog No 3471) at a density of 10000 cells/ml and cultured in serum-free mammary epithelial basal medium media (Lonza, Walkersville, MD, Catalog No CC-3150) supplemented with 20 ng/mL EGF (Chemicon, Catalog No EA140), 5 μg/mL insulin (Sigma-Aldrich, Catalog No 15500), 1 μg/mL hydrocortisone (Stem Cell Technologies, Catalog No 07904), 20 ng/mL bFGF (Invitrogen; Catalog No 13256), B27 (Invitrogen; Catalog No 17504), 4 μg/mL hePARin (Stem Cell Technologies, Catalog No 07980), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Stem Cell Technologies, Catalog No 07500). Cells were fed every three days by adding additional media to wells. After 7days, diameters and numbers of aggregated cell spheres were measured using Celigo Cytometer (Cyntellect, San Diego, CA).
8
Culturing Various Human Cell Lines
9
Culturing Human Cell Lines for Biomedical Research
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HEK293 and adult normal human dermal fibroblasts (NHDFs) were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin, and 100 μg/ml glutamine (Thermo Fisher). M2-10B4 and S1/S1 stromal cells were obtained from Stem Cell Technologies and maintained in DMEM with 10% FBS and penicillin, streptomycin, and glutamine as previously described (87 (link)). CD34+ hematopoietic progenitor cells (HPCs) were isolated from deidentified human fetal liver obtained from Advanced Bioscience Resources as previously described (88 (link)). All cells were maintained at 37°C and 5% CO2.
10
Culturing Human and Mouse Immune Cells
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