1、Cellular subculture and the effect of MTT assay of scanning of peroxide to the growth and survival of cells in vivo.Hu Boqiang1()Dong ZhiWei11()1School of Life Science, Beijing Institute of Technology, Beijing ,ChinaCellular subculture assays is the rudimental method of all cellular experiments. Via

2、 subculture, cells could survive, duplicate and preserve their own genes. In our experiment, we transferred the subculture cells to 96-well plate, treating with H2O2 in different concentrations and dying living cells (6251 cell line) with MTA. Finally, we got the effect of H2O2 in different concentr

3、ations to the surviving and growth of 6251 cells.1. IntroductionHydrogen peroxide (H2O2) is the simplest peroxide (a compound with an oxygen-oxygen single bond). It can generate inside the cell. Furthermore, it is also a strong oxidizer (According to Wikipedia, item Hydrogen peroxide) which can fata

4、l to the survival of Eukaryotic cells. However, thanks to Catalase (CAT), the enzyme which could reduce H2O2, cells could survival in vivo.Although CAT could reduce the side effect of H2O2, excessive concentration of H2O2 can cause serious diseases to people, especially in aged groups whose CAT acti

5、vity were lower than the young ones. Paradoxically, H2O2 plays a “double-edge blade” role in the pathology of cancer. On the one hand, it can intervene cellular DNA repairment, intracellular signals transfer pathway, the adherence of the cell and promoting the expression of VEGF, which could acceler

6、ate the spread of tumor. On the other hand, however, H2O2, the indicator of Oxidative Stress, can induce to cell apoptosis and depress the duplication of cells. So doctors usually take advantage of H2O2, like Radiotherapy (using Ultraviolent to produce H2O2 and other peroxide) to try to cure cancer

7、(Yao&Zhong, 2006).Peroxide can be found everywhere in daily life. For example, in hair dye, peroxide can make hair more flexible by cutting up the disulfide bonds between different keratin chains. As we do not know whether peroxide would be harmful and, if not harmful, what is the maximum concentrat

8、ion of peroxide for human cells, an experiment is required to examine that.MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a yellow tetrazole), is reduced to purple formazan in living cells. A solubilization solution is added to dissolve the insoluble purple formazan product int

9、o a colored solution. The absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer, and because the absorbance shows significant relevant to the number of living cells, it is a good way to count the number of t

10、hem. A main application allows assessing the viability (cell counting) and the proliferation of cells (cell culture assays). It can also be used to determine cytotoxicity of potential medicinal agents and toxic materials, since those agents would stimulate or inhibit cell viability and growth. This

11、assay would be used in this experiment to detect the effect of peroxide to 6251 cells. (According to Wikipedia, item “MTT Assay”)2 The process of the experimentInstrument: Micro plate reader, inverted microscope, countering plate, 96-well plate, Pipette (Eppendorf).Material: 6251 cell lines in vitro

12、.Reagent: 2mg/ml MTT, trypsin, PBS buffer, FCS-PBS, DMSO.Steps: 1. Inoculation of cell. Take the plastic culture plate from insulation can to the air clean bench. Wipe the plate with Ethanol. After all ethanol evaporate, set the plate on the alcohol burner to kill bacteria on the surface of the plat

13、e. Open the plate, then remove all nutrient fluid off the plate and add 1ml trypsin in the plate. After 5 minute, if all cells become round and flow with the liquid (see in the microscope), remove all trypsin solution and add 2ml nutrient fluid. Flush the bottom of the plate for at least 5mins, to m

14、ake sure all cells are movable. Then suck a single drop of the liquid to count the number of cells.Add cellular solutions and buffer to the 96-well plate (as described in the table below), then culture the cells for26 hours in 37 to make the cells paste on the wall of the wells.H1GFEDCBA2150ul nutri

15、ent fluid150ul nutrient fluid150ul nutrient fluid3150ul cellular solution150ul cellular solution150ul cellular solution4150ul cellular solution150ul cellular solution150ul cellular solution5150ul cellular solution150ul cellular solution150ul cellular solution6100ul cellular solution100ul cellular so

16、lution100ul cellular solution789101112 2. Adding drugs: removing all liquid in row 3-6 of line GEF. Add 100ul 100umol/mlH2O2 in row 3, 200umol/mlH2O2 in row 4 and 500umol/mlH2O2 in row 5 (We first added 150ul in all wells, and after 30min all H2O2 were removed and added 100ul H2O2 solution instead,

17、which may be the most important reason for the high death rate in our finally result).3. Washing the plate with 200ul 3% FCS-PBS for each well. Be careful to incline the plate to make it easy to suck the liquid and DO NOT let the pipette prick the bottom of the well.4. Solute all cells with DMSO, 20

18、0ul for each well. Keep shaking the plate for 30min and the solution in the plate would turn purple.5. Put the plate into the Micro plate reader and read OD550.3. Result3.1 The Number of living cell53 44Table 1Number for living cells = （living cells in 4 corners）4*104=4104 /mlAs the number of living

19、 cells is suitable for following steps, we did not dilute the solution.3.2 Survival rate after treated with peroxideSurvival rate = （Row Average-Empty Control）（Cell control-Empty Control）Empty Control100umol/mlH2O2200umol/mlH2O2500umol/mlH2O2Cell controlE0.0730.0820.0660.0710.288 F0.0690.0720.0610.0

20、680.668G0.0860.1140.0790.1010.427Average0.0760.0890.0690.0800.692*Survival rate2.2%-1.2%0.6%Table 2 （6251 cell）*（As there are only 100ul in cell control group while in others there are 150ul, the data in last line were multiplied with 1.5 to get the final result in calculation of survival rate）H2O2

21、concentration: umol/LFigure 1 (6251 cell)3. Conclusion and DiscussionThe data in row 4, treated with 200umol/L H2O2, is questionable: we even get negative survival rate. Another question is there are so many cells died that the result in our experiment is too low to be persuasive. Besides, date in l

22、ine G is significantly higher than that in line E or F, except row Cell control. Maybe it is because of marginal effect. Comparing with others published works (Yao, 2006 & Duan, 2010) which succeed in finding the relationship between the concentration of H2O2 and the survival rate of the cells, we f

23、ind that, they used H2O2 in lower concentration. The range of our data is from 100umol/L to 500umol/L while other researchers used 50-150umol/L or 10-50umol/L. So besides the mistakes we made during the experiment, high concentration of peroxide is another reason for the failure of the experiment. Furthermore; comparing our data with those published ones (figure 2), we find