Primary cell passage technology
First, the digestive method of adherent cells
1. Aspirate or pour off the old culture solution in the bottle.
2. Add about 1 ml of digestive juice (trypsin or mixed with EDTA) and gently shake the flask so that the bottom cells are immersed in the solution.
3. After 2-5 minutes of digestion, the culture flask was placed under a microscope to observe that the cells adhering to the original wall gradually became round. When the float was not floated, the digestive juice was discarded, and the culture solution was added to terminate the digestion.
4. Blot the adherent cells into a suspension with a pipette, inoculate them into two or three additional flasks, and continue to culture in a 37 °C incubator. The growth of the adherent wall was observed the next day.
Second, the passage of suspended cells
1. Direct passage 1 After the suspended cells are slowly precipitated at the bottom of the bottle, the supernatant is sucked out by 1/2-2/3.
2 After pipetting to form a cell suspension, inoculate them into two or three additional flasks and incubate them in a 37 °C incubator.
2. Centrifugation Passage 1 Transfer the cells together with the culture solution to a centrifuge tube and centrifuge at 800-1000 rpm for 5 minutes.
2 Remove the supernatant, add new culture solution to the centrifuge tube, and blow with a pipette to form a cell suspension.
3 Inoculate the cell suspension into two or three additional flasks and incubate in a 37 ° C incubator.
Primary cell cryopreservation
1. Select primary cells with good growth and a large number of cells, and change the solution one day before cryopreservation.
2. The adherent cells need to be digested with 0.25% trypsin to digest the cells, and the cell suspension is collected into a centrifuge tube.
3. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant.
4. Precipitate the culture medium containing DMSO, count, and adjust to (1-10)×106/ml.
5. Dispense the suspension into a cryotube, 1 ml per tube.
6, seal the frozen tube, the seal must be strict, otherwise it will be prone to burst when resuscitation.
7. Mark the cell type with a marker and freeze the date.
8. Decrease in the following order: room temperature → 4 ° C (20 minutes) → refrigerator freezer (30 minutes) → ultra-low temperature refrigerator (-80 ° C overnight) → liquid nitrogen.
Primary cell resuscitation technique
1. Remove the cells and quickly thaw them in warm water at 37 °C.
2. Aspirate the cell suspension and add 10 times more culture solution.
3. Centrifuge for 5 minutes at 1000 r/min and remove the supernatant.
4. After appropriately diluting with the culture solution, inoculate the culture flask and place it in a 37 ° C CO2 incubator for static culture.
Microscopic examination of cell adhesion ability. The culture medium was changed the next day and the culture was continued.
Primary cell staining technique
one. Trypan blue staining
1. Prepare a single cell suspension and properly dilute (106 cells/ml).
2, staining: Take 9 drops of cell suspension into a small test tube, add a drop of 0.4% trypan blue solution, mix.
3. Counting: In 3 minutes, live cells and dead cells were counted separately using a blood cell counting plate.
4. Under the microscope, dead cells are stained blue, while living cells are rejected.
5. Calculate cell viability according to the formula.
two. Eosin Y staining
1. Prepare a cell suspension of 1 x 106-5 x 106 cells/ml.
2. Mix 0.1 ml of cell suspension with 0.1 ml of Eosin Y stain.
3. Count the number of live and dead cells under the microscope.
4, under the microscope, the red is dead cells. Calculate cell viability according to the formula.
three. Aniline black staining experiment
1. Prepare a cell suspension of 1 x 106-5 x 106 cells/ml.
2. Mix 1 part of 1% aniline black solution with serum containing physiological saline 1:9 to make 0.1% aniline black dye solution.
3. Mix 0.1 ml of cell suspension with 0.1 ml of aniline black stain. Leave at room temperature for 10 minutes.
4. Count with a counting plate, black cells are dead cells, and calculate the living cell rate according to the formula.
Primary cell counting technique
1. Prepare the counting plate: Clean the counting plate and special cover glass with alcohol, then gently dry.
2. Preparation of cell suspension: Disperse monolayer cultured cells with digestive juice or directly collect suspension cultured cells to prepare a single cell suspension. The cell density is required to be not less than 104/ml. If the number of cells is small, the suspension should be centrifuged (1000 rpm, 2 minutes) and resuspended in a small amount of the culture solution.
3, loading: use the customary light to blow the cell suspension, take a little cell suspension, add a small amount of cell suspension on the side of the cover glass on the counting plate.
4. Counting: Calculate the total number of cells in the four large cells of the counting plate. The pressure line cells only count the left side and the upper side. Then calculate according to the following formula: cell number / ml = 4 large cells total / 4 × 10000.
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