Operating Procedures for Embryonic Stem Cell Culture

Operating Procedures for Embryonic Stem Cell Culture

different size petri dish,dish petri,disposable sterile petri dish,15cm petri dish,sterile plastic petri dishes ]
Maintain ES cells in an undifferentiated state: ES cell culture uses high-sugar medium containing ESGRO (leukemia inhibitory factor) to prevent cell differentiation. Provide cells with a plate coated with 0.1% gelatin as a matrix for cell adhesion. It is recommended that the cells are passaged at a ratio of 1:8 every 2-3 days from the plate that has reached 80%-90% confluence. After the cells are passaged, the cells should be seeded in advance before inoculating the cells in a 0.1% gelatin-coated culture dish. On the uncoated tissue culture plate for 2 hours, the differentiated cells were allowed to adhere to separate the differentiated and undifferentiated cells. The cells were cultured at 37°C, 5% CO2, and 100% humidity.[ different size petri dish,dish petri,disposable sterile petri dish,15cm petri dish,sterile plastic petri dishes ]
Medium: ES: prepare a 20× solution without DMEM, HS, ESGRO. Aliquot into 50ml FALCON tubes (diluted to 2×, 42ml per tube), and store at -20°C. The culture medium was prepared by adding 21 ml of this solution, HS and ESGRO to 450 ml DMEM, and filtered through a 0.2 μm filter. Store at 4°C.
Note: One bottle of DMEM is 500ml.[ different size petri dish,dish petri,disposable sterile petri dish,15cm petri dish,sterile plastic petri dishes ]
Stock solution: DMEM (high glucose), horse serum (HS), L-glutamine (200mM), MEM NEAA (10mM), HEPES (1M), β-mercaptoethanol (55Mm), PEST, ESGRO.
Resuscitating cells: Cells are frozen in 10% dimethyl sulfoxide (DMSO) to prevent the formation of crystals, which can damage the cells. However, dimethyl sulfoxide is toxic to cells, and rapid cell recovery is very important.
Steps:
1. Take out a tube of cells from liquid nitrogen;
2. Place the cryopreservation tube in a 37°C water bath for 2 minutes (or put the solution in the tube to just completely dissolve);
3. Transfer the cells to a 15ml Falcon tube;
4. Add 5ml ES medium (flush the cryotube with medium);
5. Centrifuge for 3 minutes;
6. Discard the supernatant, resuspend the cells in 2ml ES medium and pipette at least 10 times;
7. Inoculate in a 6-well or 6cm tissue culture dish coated with gelatin (see below);
8. Incubate.
Cryopreserved cells
Cryopreservation solution: 90% HS and 10% dimethyl sulfoxide
step:
1. Wash the cells with 1×PBS and leave a little PBS in the petri dish;
2. Collect the cells with a cell scraper;
3. Transfer the cells into a 15ml Falcon tube and centrifuge for 3 minutes;
4. Discard the supernatant and resuspend the cells in cold cryopreservation solution (2ml for 10cm petri dishes, 6-7ml for 15cm petri dishes.)
5. Dispense into cryopreservation tubes, 1ml per tube;
6. Set it at -80°C overnight, and transfer to liquid nitrogen the next day.
Gelatin coating: prepare 500ml 0.1% gelatin solution
1. Dissolve 0.5g gelatin in 500ml calcium and magnesium-free PBS (50-65℃ water bath for 15-30 minutes).
2. It is best to filter the solution through a 0.2μm membrane without cooling and store it at 4°C.
Coated culture plate or petri dish .
1. Add enough gelatin solution to cover the culture surface (15cm petri dish plus 2ml, 10cm petri dish plus 0.5-1ml, the amount of solution is not important, as long as it can completely cover the culture surface.);
2. Put it at room temperature for 30 minutes;
3. Remove the gelatin solution and store the culture plate in a packaging bag at room temperature. It is best to square the plate to prevent gelatin from contaminating the lid and flowing out of the culture plate.[ different size petri dish,dish petri,disposable sterile petri dish,15cm petri dish,sterile plastic petri dishes ]

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Post time: Nov-15-2021

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