细胞生物化学实验课件Exp 7-cell culture and cell fusion-Furong Gao
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2. Dilute chick blood red cells and adjust cell concentration; 3. Calculate 0.5 g PEG , heat on the alcohol burner to melt it, then cool down and
add pre-warmed 0.5 mL Hanks solution, leave at 37℃; 4. Mix well of the Hela cells and the chick blood red cells, centrifuge at 1000
【Principle】
1. Cell fusion naturally happened during life evolution, we can use PEG (polyethylene glycol, MW=200~6000. 1000 is better) as the mediator to aid the fusion of human Hela cells and Rat blood red cells.
medium ( time course for option); 7. Take some solution for observation of cell fusion under the microscope.
【Results and analysis】
Probable fused cells
✓ To grasp the cell fusion technique by performing the cell fusion experiment
【Principle】
1. When the cell is not used for experimental analysis, it is usually stored in liquid nitrogen. Dimethyl sulfoxide (DMSO) is a small molecular and easy-soluble protector when cell suffers freezing.
2. Dislodge the cells by trypsin; 3. Dilute the cells with growth medium; 4. Transfer the cell suspension to a 15 ml conical tube, centrifuge
at 500g for 5 mins at RT and remove the growth medium by aspiration; 5. Resuspend the cells in 1-2ml of freezing medium; 6. Transfer the cells to cryovials, incubate the cryovials at -80 C overnight 7. Next day transfer the cryovials to Liquid nitrogen.
2. Please put on your lab coat, wear mask and gloves; 3. Keep yourself from any dangers in the lab; 4. Get a well organized team work when doing experiment.
Process of animal cell fusion
Cell membrane linked
Notes
1. Please read the experimental book to get familiar with what you will do in the lab doing experiment;
【Principle】
1. Cell counting with hemacytometer to determine the concentration of cells. We use a graved space with a certain liquid volume to determine how many cells are included there.
(For adherent cells, start from step 1, suspension cells from step 4)
【Procedure】
Cell fusion
1. Collect Hela cells by trypsin, centrifuge at 1000 rpm/5 min and discard the supernatant and resuspend in Hanks solution,;
3. 10% DMSO and 10% fetal bovine serum (FBS) in basal medium can serve as a good cell freezing medium. But at room temperature, DMSO is harmful to cells. So, when thawing cells, we must immediately put the cells into 37 ℃ to let the cells quickly thawed and get rid of DMSO.
细胞生物化学实验课件Exp 7-cell culture and cell fusion-Furong Gao
Overview
• Objective • Principle • Procedure • Results and analysis • Notes • Thought questions
2. Transfer the thawed cells into the ready centrifuge tube and Centrifuge at 1000 rpm/min for 5 min and discard the supernatant.
3. Re-suspend the cells with 5 mL fresh culture medium and leave 0.5 mL for cell viability determination and the residue was added into the flask for culturing at 37℃, with 5% CO2.
【Thought questions】
1. What is the principle of cell fusion?
2. How cell fusion is applied in monoclonal antibody ?
3. Analyze your results and put forward any questions and problems that may interrupt your results during experiment.
2. To protect the cells from breaking up when meeting sudden decrease of temperature, it is necessary to put cells in freezing container and immediately into first at -20 ℃ then at -80 ℃ at last into liquid nitrogen at -196 ℃. In contrast, when thawing cells, we must immediately put the cells into 37 ℃ to let the cells quickly thawed and get rid of DMSO. (Slow freezing Fast thawing)
1 ×10-4 mL
【Principle】
2. Trypan blue is a cell dye which is usually used to distinct live cells from dead cells. The mechanism is that the live cells can exclude this dye from their invasion into their intact cell membrane while the dead cells with broken cell membrane fail to do that. So dead cells will be stained blue while the live cells keep bright.
rpm/5 min, discard the supernatant; 5. Add the prepared PEG dropwise into the above mixed cells and leave at 37℃
for 5 min; 6. Centrifuge and discard the supernatant then resuspend the cells in RPMI1640
2. The mediator PEG can decrease the free water between cells and destroy the phospholipid bilayer of cell membrane. The cell membrane structure is changed and make cells easier to contact and come into one.
???Βιβλιοθήκη 【Procedure】Cell thawing
1. Take one tube of cells from the liquid nitrogen tan and immediately put it into the water bath. Shake softly to make it thaw quickly.
【Objective】
✓ To learn basic techniques of cell culture and cell viability measurement and cell counting;
✓ To understand the principle of cell fusion induced by PEG;
4. Change the medium on the next day.
【Procedure】
Cell freezing
1. Remove the growth medium, wash the cells by PBS and remove the PBS by aspiration; (Why wash with PBS?)
add pre-warmed 0.5 mL Hanks solution, leave at 37℃; 4. Mix well of the Hela cells and the chick blood red cells, centrifuge at 1000
【Principle】
1. Cell fusion naturally happened during life evolution, we can use PEG (polyethylene glycol, MW=200~6000. 1000 is better) as the mediator to aid the fusion of human Hela cells and Rat blood red cells.
medium ( time course for option); 7. Take some solution for observation of cell fusion under the microscope.
【Results and analysis】
Probable fused cells
✓ To grasp the cell fusion technique by performing the cell fusion experiment
【Principle】
1. When the cell is not used for experimental analysis, it is usually stored in liquid nitrogen. Dimethyl sulfoxide (DMSO) is a small molecular and easy-soluble protector when cell suffers freezing.
2. Dislodge the cells by trypsin; 3. Dilute the cells with growth medium; 4. Transfer the cell suspension to a 15 ml conical tube, centrifuge
at 500g for 5 mins at RT and remove the growth medium by aspiration; 5. Resuspend the cells in 1-2ml of freezing medium; 6. Transfer the cells to cryovials, incubate the cryovials at -80 C overnight 7. Next day transfer the cryovials to Liquid nitrogen.
2. Please put on your lab coat, wear mask and gloves; 3. Keep yourself from any dangers in the lab; 4. Get a well organized team work when doing experiment.
Process of animal cell fusion
Cell membrane linked
Notes
1. Please read the experimental book to get familiar with what you will do in the lab doing experiment;
【Principle】
1. Cell counting with hemacytometer to determine the concentration of cells. We use a graved space with a certain liquid volume to determine how many cells are included there.
(For adherent cells, start from step 1, suspension cells from step 4)
【Procedure】
Cell fusion
1. Collect Hela cells by trypsin, centrifuge at 1000 rpm/5 min and discard the supernatant and resuspend in Hanks solution,;
3. 10% DMSO and 10% fetal bovine serum (FBS) in basal medium can serve as a good cell freezing medium. But at room temperature, DMSO is harmful to cells. So, when thawing cells, we must immediately put the cells into 37 ℃ to let the cells quickly thawed and get rid of DMSO.
细胞生物化学实验课件Exp 7-cell culture and cell fusion-Furong Gao
Overview
• Objective • Principle • Procedure • Results and analysis • Notes • Thought questions
2. Transfer the thawed cells into the ready centrifuge tube and Centrifuge at 1000 rpm/min for 5 min and discard the supernatant.
3. Re-suspend the cells with 5 mL fresh culture medium and leave 0.5 mL for cell viability determination and the residue was added into the flask for culturing at 37℃, with 5% CO2.
【Thought questions】
1. What is the principle of cell fusion?
2. How cell fusion is applied in monoclonal antibody ?
3. Analyze your results and put forward any questions and problems that may interrupt your results during experiment.
2. To protect the cells from breaking up when meeting sudden decrease of temperature, it is necessary to put cells in freezing container and immediately into first at -20 ℃ then at -80 ℃ at last into liquid nitrogen at -196 ℃. In contrast, when thawing cells, we must immediately put the cells into 37 ℃ to let the cells quickly thawed and get rid of DMSO. (Slow freezing Fast thawing)
1 ×10-4 mL
【Principle】
2. Trypan blue is a cell dye which is usually used to distinct live cells from dead cells. The mechanism is that the live cells can exclude this dye from their invasion into their intact cell membrane while the dead cells with broken cell membrane fail to do that. So dead cells will be stained blue while the live cells keep bright.
rpm/5 min, discard the supernatant; 5. Add the prepared PEG dropwise into the above mixed cells and leave at 37℃
for 5 min; 6. Centrifuge and discard the supernatant then resuspend the cells in RPMI1640
2. The mediator PEG can decrease the free water between cells and destroy the phospholipid bilayer of cell membrane. The cell membrane structure is changed and make cells easier to contact and come into one.
???Βιβλιοθήκη 【Procedure】Cell thawing
1. Take one tube of cells from the liquid nitrogen tan and immediately put it into the water bath. Shake softly to make it thaw quickly.
【Objective】
✓ To learn basic techniques of cell culture and cell viability measurement and cell counting;
✓ To understand the principle of cell fusion induced by PEG;
4. Change the medium on the next day.
【Procedure】
Cell freezing
1. Remove the growth medium, wash the cells by PBS and remove the PBS by aspiration; (Why wash with PBS?)