Embedded Files
The purpose of this lab is to learn basic tissue culture techniques and learn the procedure of subculturing cells and counting them.
Materials required
HEK 293 Cell Line (optimal 70% confluency)
500 ml of inimum Essential Media
Trpsin/EDTA aliquoted in 15 ml tube
Four 10-cm tissue culture plates
Pipet aid and some 5,10,25 ml pipets
Vacuum system and pasteur pipets
Water bath at 37 degree C
Biosafety II Hood
Olympus CK2 inverted Phase Microscope with digital camera
Stacked Incubators
Clinical Centrifuge
Protocol
Biosafety II hood was turned on with UV lights and fan while the sash is down
Water bath at 37 degree C was ready to use
After turning those on and washing hands thoroughly, a 500 ml MEM bottle and 15 ml tube of Trypsin/EDTA were placed into water bath for 15 minutes to warm up.
A plate of HEK 293 cells was selected to check their confluency under the CK2 inverted phase microscope. Select the best plate with confluency between 40-85%.
After 15 minutes, UV lamp in biosafety hood was turned off and the sash was raised upto the marked area and regular lights were turned on. The area was cleaned with 70% Ethanol and media bottle and TE tube were thoroughly rinsed with 70% ethanol before placing them in the hood.
The selected plate of cells were transferred to biosafety hood.
Vacuum system was turned on and pasteur pipet was attached to it, which is used to suck media off of cells.
4 ml of TE was added to the cells and incubate for 5-10 minutes in 37 degree C incubator.
While cells are incubating, 10 ml media was added in a 15 ml tube and in four 10-cm plates.
The cells were gently swirled and checked with microscope. All the trypsinized cells were then transfered to a 15 ml tube containing 10 ml of MEM media.
Cells were then centrifuged at 30 speed for 3 minutes in the clinical centrifuge.
Supernatant was carefully sucked off with pasteur pipet and the cell pellet was resuspended by tituration with 8 ml of MEM.
The cells were immediately split 1 to 4. 2 ml each of cells was transferred to two new 10-cm plates, which already has 10 ml of ME. Each plate now had 12 ml each total.
The remaining 4ml of cells in the tube were mixed and counted 20 ul of cells using hemocytometer. 20ul of cells from the 15 ml tube was added to the wedge under the edge of coverslip on the hemocytometer.
The cells were counted using hemocytometer in the microscope. The calculations were as follow:
After counting all four sections(16 squares/section), add the counts for all four sections and divide by four. Take this average and multiply by 10^4 to get number of cells per ml. The total number of cells in 15 ml tube is 4 times cells/ml of media. Add 500,000 cells per plate, 500,000 divided by cells/ml.
Calculations:
First trial:
56 + 34 + 32 + 38 = 160 cells
160/ 4 = 40 average
40 x 10^4 = 400,000 cells per ml of media
500,000 cells per plate divided by 400,000 cells/ml = 1.25 ml per plate = 1250 ul per plate
Second trial:
42 + 33 + 58 + 67 = 200
200/4 = 50
50 x 10^4 = 500,000 cells per ml
to add 500,000 cells,
500,000 cells/500,000 cells per ml = 1 ml per plate = 1000 ul per plate
Based on the calculations, 500,000 cells were transferred to the two remaining tissue plates containing 10 ml of MEM that have not yet recieved any cells. The cells plates were placed back in the incubator. Everything was cleaned with 70% ethanol and any equipment used were turned off.
Following day,cells were inspected for adhesion using inverted microscope. Although cells areexpected to not ahere until after day 2.
On day three, after trypsinization, photographs of cells were taken using the Olympus inverted phase microscope with the digital camera.
The process of microphotography is as follows as mentioned in the protocol:
Select the best plate of cells. Turn on microscope and computer. Open the infinity program on the computer. Adjust the brightness of the lamp for the best view, adjust the binocular eyepiece and then focus the cells to capture picture. Use the 10X, 20X, and 40X magnification on the microscope to capture the best picture of your cells.
On day four, when one of the plates has cells at around 70% confluency, cells were split 1 to 8 into a new tissue culture plate. They were resuspended with 8 ml of media after trypsinization and 1 ml of cells were transferred in a 10-cm plate containing 10 ml of MEM. Thus the final volume came to be 11 ml in the plate. the remaining cells were discarded into biohazard bag. Cells were split every five to seven days to maintain a regular supply of cell culture.
Results
Experiment was needed to be repeated with the same steps as mentioned above because of the contamination in cell plates.
Second trial of this lab had good results and all plates were at good confluency. There were no contamination in any of the plates.
In 1-2 days, check the adherence and the confluency of the trypsinized cells and split them.
Conclusion
First trial of this lab was not successful because of contamination of plates.
The possible reason for the contamination could be pipet tip touching the outside of bottle/tube, or glove touched the tissue culture plate while transferring cells.
Second trial of this lab was successful. All plates had good confluency and there were no contamination.
Cells were regularly split to maintain a regular supply of HEK 293 cells
Page updated
Google Sites
Report abuse