Flash Link: https://vlab.amrita.edu/repo/BIOTECH/CEL/Isolation_of_Endoplasmic_racticulum/index.swf
Materials Required
1. T150 cell culture flask
2. DMEM cell culture medium
3. 1x PBS buffer
4. 1x isotonic extraction buffer (10mM HEPES, pH 7.8, 250mM sucrose, 25mM potassium chloride, and 1mM EGTA)
5. Trypsin /EDTA solution
6. Cooling centrifuge
7. Dounce homogenizer
8. Inverted microscope
9. Ice bucket
10. Suction pump
11. Ultracentrifuge
12. CO2 incubator
13. 50 ml centrifuge tubes
14. 1.5 ml micro centrifuge tubes.
Procedure
1. Take out a T150 flask containing the adherent (HeLa) cells (2x 108) from the carbon dioxide incubator.
2. Remove the media from the T150 flask completely and add 20 ml of 1x PBS.
3. Remove the PBS and add 4ml trypsin/EDTA solution to the flask and incubate for 2 minutes in a carbon dioxide incubator.
4. Observe the cells under the phase contrast microscope to check the detachment of cells.
5. Add 16 ml cell culture media into the flask and mix gently.
6. Transfer the entire contents into a 50 ml centrifuge tube and centrifuge at 1000 rpm for 4 minutes.
7. Remove the supernatant and wash the cell pellet once with 20ml 1xPBS.
8. Centrifuge again at 1000rpm for 4minutes and add 1.2 ml of 1xPBS to the cell pellet
9. Transfer the contents into a 1.5 ml microfuge tube and centrifuge at 1000rpm for 4 minutes.
10. Discard the supernatant and keep the cell pellet in ice.
11. Homogenize the cells with 1.2 ml of pre cooled 1x extraction buffer in a dounce homogenizer .The dounce homogenizer should be rinsed in pre cooled 1x extraction buffer before homogenization.
12. Give 10-20 strokes with the dounce homogenizer.
13. Check the cell lysis on a glass slide with cover slip till 70-80 % cells are broken.
14. Observe under the microscope to check whether the cells are broken. Lysed cells look pale in color (in a glass slide).
15. Transfer the homogenized cells into a 1.5 ml precooled microfuge tube and Centrifuge at 4degree C for 10 minutes at 2500 rpm
16. Collect the supernatant in a fresh tube and centrifuge at 14,000rpm for 15 minutes.
17. Collect the supernatant in a fresh 1.5 ml ultracentrifuge tube and Centrifuge at 43000 rpm for 1 hr at 4 degree Celsius in an ultracentrifuge.
18. Discard the supernatant and store the cell pellet in 1x extraction buffer in -20 degree. Colorless pellet will contain the crude microsomes.
Difference Encountered in a Real Laboratory
In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab:
1. Before starting the experiment sterile the laminar air flow chamber using spirit.
2. All the tubes and bottles used in the experiment should be labeled properly.
3. Always disinfect your work area when you are finished.
