BT474 cells were transfected and seeded into 96-well plates (6×103 cells per well) for the proliferation activity which was determined at 0, 6, 12, 18, and 24 h post-transfection using the Cell Counting Kit-8 (Dojindo, Japan) according to the manufacturer’s instructions. For the migration assay, transfected BT474 cells were treated with mitomycin C (10μg/ml, R&D Systems, USA) for 10 h to prevent proliferation, and equal number of cells were then seeded into the 24-Well Millicell plates (Millipore, Germany) containing an 8-μm pore membrane as previously described [14 (link)] and the transwell-containing plates were incubated for 24 h.
Mitomycin c
Manufactured by R&D Systems
Sourced in United States
Mitomycin C is a laboratory reagent used for research purposes. It is a DNA-damaging agent that inhibits DNA synthesis and cell division. Mitomycin C is commonly used in cell biology and cancer research studies.
Automatically generated - may contain errors
Lab products found in correlation
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Diff quik, by Sysmex (1 mentions)
Bx71 microscope, by Olympus (1 mentions)
Sodium pyruvate l glutamine, by Avantor (1 mentions)
Ultralow igg hi fbs, by Thermo Fisher Scientific (1 mentions)
Mrc 5, by American Type Culture Collection (1 mentions)
Thiazolyl blue tetrazolium bromide, by Merck Group (1 mentions)
Phloretin, by Merck Group (1 mentions)
Prism 6, by GraphPad (1 mentions)
Mouse ifn γ development module, by R&D Systems (1 mentions)
Elispot blue color module, by R&D Systems (1 mentions)
Mitomycin c, by Nacalai Tesque (1 mentions)
7 protocols using mitomycin c
1
Proliferation and Migration Assays of BT474 Cells
2
Wound Healing Assay with miR-29b and HSP47
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The A549 cells were seeded into 6-well cell culture plates. The cell culture was allowed to reach approximately 90% confluence, and then placed for 24 h in a serum-free RPMI-1640 medium for serum starvation. Next, the cells were treated with mitomycin C (5 µg/mL; R&D Systems, Minneapolis, MN, USA) for 15 min to prevent cell proliferation. A sterile 200-µL yellow pipette tip (Labcon, Petaluma, CA, USA) was used to make a straight scratch. Cell debris was washed out with the serum-free media and was further transfected with miR-29b mimic or siHSP47 before treatment with TGF-β1 for 72 h. The cells were stained with Diff-Quik (Sysmex, Kobe, Japan), and images of the migrated cells were acquired using an Olympus BX71 microscope (Olympus, Tokyo, Japan). In the micrograph, the area in between the lines refers to the area of the initial wound scratch, and cells identified between the scratch lines were counted with ImageJ analyzer (National Institutes of Health [NIH], Bethesda, MD, USA).
3
Desialylation and Glycophenotyping of Cells
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The cells (1 × 106 cells/mL) were pre-treated with 100 ng mL−1 of mitomycin C (R&D, 3258/10) in serum-free medium for 3 h at 37 °C, washed and resuspended, and then desialylated with 100 mU mL−1 of α2-3 and α2-6 neuraminidases (NAs) from Clostridium perfringens (Sigma, N2876) and Arthrobacter ureafaciens (Roche, 10269611001) in the same medium for 30 min at 37 °C, respectively. The terminal α2,3/α2,6 sialic acid and Gal/GalNAc residues on the resultant cells were identified using flow cytometry (FCM) with Biotinylated MALII/DyLight 488 streptavidin (B-1265-1/SA-5488-1), Fluorescein labeled SNA (FL-1301), and Cy5 labeled PNA (CL-1075-1).
4
MRC-5 Fibroblast Cell Preparation
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Human fibroblast cell line MRC-5 (CCL-171) was obtained from ATCC® (Manassas, VA) and grown in B cell growth media containing Corning® DMEM [+] 4.5 g/L glucose, sodium pyruvate [–] L-glutamine (VWR International, Radnor, PA), 1xPen/Strep/Glu and 10% ultralow IgG HI-FBS (Thermo Fisher Scientific, Waltham, MA), to 80% confluence before being treated for 4 h with 5 μg/ml mitomycin C (Tocris, R&D Systems). Monolayers of growth arrested cells were washed 3 times with PBS, harvested with trypsin, neutralized with growth media, washed 1x in growth media and finally cryopreserved using 10% DMSO, 30% HI-FBS in growth media.
5
Cytotoxicity Assay of Chemoagents
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Cells were plated in 96 well plates and allowed to reach confluency (approximately 1 day) to enter the plateau phase of growth, mimicking the non-proliferating state of PMP in vivo, and the media replaced. Cells were then exposed to a dose range of Mitomycin C, Oxaliplatin (R&D Systems, Abingdon, UK), or Phloretin (Sigma Aldrich) for 60 minutes at 37°C. Media was then replaced and the cells cultured under normal conditions for a further 4 days. Cellular metabolism was assessed using the MTT assay by addition of 1/10 volume (10 μl) of 5 mg/ml Thiazolyl Blue Tetrazolium Bromide (Sigma Aldrich) and incubation for 4 h. 75 μl of media was then removed and 50 μl DMSO added to solubilize the MTT crystals. Plates were incubated at 37°C for 10 minutes and then absorbance measured at 540 nm. Growth curves were fitted using a four parameter sigmoidal algorithm in GraphPad Prism 6 (GraphPad Software Inc. La Jolla, CA, USA) and IC50 values derived.
6
Mitomycin C Pretreatment of Caco-2 Cells
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Mitomycin C (R&D Systems 3258/10) was dissolved in DMSO to 10 mg/mL and stored at −20°C until time of experiments. It maintained effectiveness for up to 6 months at −20°C. For Mitomycin C experiments in Figure 4 , Caco-2 cells were grown to 95% confluence and treated with 10 μg/mL Mitomycin C-containing media for 2-3 hours. After 2 PBS washes, cells were given normal media again until 3-4 days post-MMC treatment. At that point, cells were infected.
7
Quantifying Tumor-Specific T-Cell Response
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Spleens from DC–tumor cell fusion vaccine‐treated mice were isolated 10 days after the last DC–tumor cell fusion vaccine injection. Splenocytes were isolated from the spleens as described previously. To stimulate splenocytes, B16‐F10 melanoma cells were treated with 15 μg·mL−1 mitomycin C (Nacalai Tesque Inc.) for 45 min. Splenocytes harvested from vaccine‐treated mice and mitomycin C‐treated B16‐F10 melanoma cells were mixed at a ratio of 10 : 1 and co‐cultured for 48 h. Nonadherent splenocytes were collected, and ELISpot assay was performed using the Mouse IFN‐γ Development Module (R&D Systems, Minneapolis, MN, USA) and the ELISpot Blue Color Module (R&D Systems). The numbers of IFN‐γ‐secreting cells were subsequently counted.
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