The mouse mast cell line MC/9 was obtained from ATCC (ATCC designation CRL-8306) and was cultured in DMEM (Life Technologies) adjusted to contain 4.5 g/L glucose and 1.5 g/L sodium bicarbonate and supplemented with 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol, 10% Rat T-STIM (Becton Dickenson) and 10% fetal bovine serum.
Rat t stim
Manufactured by BD
Sourced in United States
The Rat T-STIM is a laboratory instrument designed for the stimulation and measurement of the trigeminal sensory system in rats. It is intended for use in research applications involving the study of the trigeminal nerve and its associated structures.
Automatically generated - may contain errors
Lab products found in correlation
2 mercaptoethanol, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Cell counting kit 8 (cck8), by Abcam (1 mentions)
Fetal bovine serum (fbs), by Avantor (1 mentions)
Mc 9 cells, by American Type Culture Collection (1 mentions)
96 well plate, by Corning (1 mentions)
24 well plate, by Corning (1 mentions)
Fetal bovine serum (fbs), by Euroclone (1 mentions)
L glutamine, by Euroclone (1 mentions)
5 protocols using rat t stim
1
Culture of Mouse Mast Cell Line MC/9
2
Mast Cell Histamine Release Assay
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The MC/9 mouse mast cells were gained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 0.05 mM 2-mercaptoethanol (Sigma, Seoul, Korea), 10% rat T-STIM (Becton Dickinson, Franklin Lakes, NJ, USA), 2 mM L-glutamine, and 10% fetal bovine serum (FBS). The viability from the cells was evaluated using a Cell Counting Kit-8 (BioVision, Milpitas, CA, USA). Histamine concentrations in the cell supernatant were measured using a histamine immunoassay kit (Oxford Biomedical Research, Oxford, MI, USA). The cells were pretreated with various concentration of extracts before 30 min. Then, compound 48/80 (25 µg/mL) was treated and the cells were incubated for an additional 30 min. After the reaction was stopped, the optical density was measured at 650 nm using a microplate reader (BioRad, Hercules, CA, USA).
3
Isolation and Expansion of Murine Bone Marrow Mast Cells
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Donor animals were euthanized by CO 2 asphyxiation prior to removal of marrow. Skin was dissected from legs using sterile forceps and scissors, and femur and tibias were removed. Tissue was scraped away from the bones. Epiphyses (bone ends) were cut with scissors to expose the medullary cavity. Using a 5ml syringe with a 25gauge needle, bone marrow was flushed out of each bone with PBS. The collected bone marrow was pooled from each mouse into one collection tube. This was centrifuged at 500xg for 5 min at room temperature (RT). Supernatant was removed and the pellet resuspend in culture medium consisting of modified Dulbecco's Modified Eagle's Medium (DMEM) with 4 mM L-glutamine adjusted to contain 4.5 g/L glucose and 1.5 g/L sodium bicarbonate and supplemented with recombinant murine 100ng/L SCL (Prepotech) and 100ng/L IL3 (Prepotech), 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol, 10% Rat T-STIM (Becton Dickenson) and 10% fetal bovine serum, and seeded into a flask. Following 2 days of culture, suspension cells (leaving debris and adherent cells) were transferred to a new flask with fresh culture medium. Complete differentiation of BMMC took 4-6 weeks, and the maturity of BMMC was assessed via flow cytometry (FACS).
4
MC/9 Cell Culture Protocol
5
Mast Cell Culture and Analysis
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MC/9 cells are cloned MCs derived from mouse fetal liver (ATCC, Rockville, MD, U. S. A.). The cell line was cultured in appropriate growth medium (High glucose Dulbecco's Modified Eagle's Medium -Euroclone -supplemented with 2 mM L-glutamine and 10% fetal bovine serum -Euroclone-, 0.05 mM 2-mercaptoethanol -Sigma-Aldrich-, 10% Rat T-STIM -Becton Dickinson, San Jose, CA, USA-) according to producer's specifications. MC/9 cells were cultured in suspension and maintained by the addition of fresh medium or its replacement. The cells were plated in 96well plates (Costar Corning Incorporate, NY, USA) at the density 6 Â 10 4 cells/well for toluidine blue staining, in 24-well plates (Costar Corning Incorporate) at the density 5 Â 10 5 cells/well for βhexosaminidase release assay and viability studies.
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