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Home > Technical services > Cytobiology

The principle of MTT
Yellow MTT (3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole salt) is reduced to blue/purple formazan by intracellular NADPH-oxidoreductases which present in the mitochondria of living cells. The formazan cannot pass through the plasma membrane and accumulates within the cell, the intracellular purple formazan can be solubilized by Dimethylsulfoxide (DMSO) and the concentration can be determined by optical density at 570 nm use the Spectrophotometer.

The absorption is directly proportional to the cell number which allows an accurate quantification of cell proliferation, viability, and cytotoxicity assays.

Assay procedure
1.Plate cells into 96-well tissue culture plates. In general, 5000 -10,000 cells per well according to the experimental factors tested.
2.Carry out your experiment by adding chemicals or biological agents into appropriate well. Incubate for 6 to 24 hours
3.Add 10 μl MTT Reagent.
4.Incubate for 2 to 4 hours until purple precipitate is visible.
5.Remove medium and add 100μL DMSO into each well to dissolve the formazan.
6.Leave at room temperature in the dark for 2 hours or shaking 15min.
7.Record absorbance at 570 nm.

Customer’s resource
Cell line

The principles of Flow cytometry
Flow cytometry employs instrumentation that scans single cells flowing past excitation sources in a liquid medium. The technology can provide rapid, quantitative, multiparameter analyses on single living (or dead) cells based on the measurement of visible and fluorescent light emission. Flow cytometry is a widely used method for characterizing and separating individual cells. This basic protocol focuses on: measure fluorescence intensity produced by fluorescent-labled antibodies and ligands that bind specific cell-associated molecules.

Assay Procedure
Immunofluorescence Staining
1.The Staining procedure is carried out at 4oC and dark. Appropriate controls are needed to get correct results, such as unstained control, isotype antibody stained control and positive control etc.
2.Make single cell suspension in L-15 at a concentration of 1-2x107 cells/ml
3.Add 50ul cell suspension to 5ml polystyrene tube (or 96 well V-bottom palte)
4.Add 50ul of appropriately diluted labeled antibody to the cells and mix gentlely
5.Incubate at 4oC in dark for 30 minutes
6.Wash cells by adding 2ml L-15 and centrifuge 5 minutes at 1000rpm (300xg)For 96 well plate, wash cells three times with 150ul L-15
7.Resuspend stained cell pellets in 400ul L-15 at 4oC for flow cytomery
8.Cells can be resuspended in fixation solution and stored for 1 week (optional)
9.Add 10ul propidium iodide prior to analysis to detect dead cells (optional)
10.Calibrate the FACScan by standard fluorescent beads (optional)
11.Flow Cytometry Analysis
 1)Flow cytometers are complex instruments that require a well-trained operator. Prior to use the Becton Dickinson FACScan, take a training course with an experienced user. Follow the rules posted in the working area for appropriate maintenance of the instrument.
 2)Turn on the FACScan power and wait until the indicator light changes from NOT READY to STANDBY. Restart the computer to connect with theFACScan.
 3)Open your account on the desktop and use the appropriate template for aquiring data (see reference for making correct setting).
 4)Turn fluid control valve to RUN and place sample tube on FACScan
 5)Analysis data with CELLQuest Software

Customer’s resource
1, sample: tissue, cell, whole blood
2, antibody

Cell Culture
Bluegene biotech is your premier partner in Cell Culture. Our Cell Culture Excellence program brings together a wide array of best-in-class products and services designed to instill confidence into your research process. You will reduce your time to achieve success by leveraging Bluegene’ comprehensive experience in cell cultivation and cell based assays which address various target types. Our knowledge will become an integral part of your research activities and will help you to avoid pitfalls and unsuccessful approaches. The outsourcing of cell production saves your internal resources, while allowing you to bring more projects to the starting block at the same time.

Cell Culture Categories
Mammalian Cell Culture
Primary Cell Culture
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