MTS Cell Proliferation and Cytotoxicity Detection Kit

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Chinese nameMTS Cell Proliferation and Cytotoxicity Detection Kit

English name: MTS Cell Proliferation and Cytotoxity Detection Kit

Product specifications: 500 times
Product Code: LZ-01X6321

Delivery cycle: 1-3 days

Product Introduction:

Experimental report:

1、 Separation and cultivation:

1. Under sterile conditions, extract atrial tissue from 1-3 day old SD rats, wash the tissue block twice with PBS, and finally cut the tissue into approximately 1mm3 size;

2. Add 4 mL of enzyme digestion solution (0.1% and 0.1% type I collagenase) to the tissue block, suspend for 10 seconds, digest at 37 ℃ for 10 minutes, then use a dropper to prepare a single-cell suspension, naturally precipitate and collect the supernatant, terminate digestion with 10% FBS medium, and place at 4 ℃;

3. Add 3-4mL of enzyme digestion solution to the remaining tissue, suspend for 10 seconds, digest at 37 ℃ for 10 minutes, collect the supernatant according to the above method, terminate digestion, and place at 4 ℃. Repeat this step 2-3 times until the tissue is digested;

4. Filter the cell digestion solution through a 200 mesh stainless steel sieve, centrifuge at 1200r/min for 10 minutes, discard the supernatant, and suspend the precipitated cells in DMEM/F12 medium containing 10% FBS. Inoculate the cells into a 25cm2 culture bottle and incubate in a 37 ℃, 5% CO2 incubator;

5. After 1 hour of differential adhesion, aspirate the culture medium and inoculate it into a 6-well plate as needed for further cultivation;

2、 Immunofluorescence identification:

1. When the atrial myocytes grow to 80% fusion, discard the culture medium and wash the cells twice with warm PBS for 10 minutes each time. Then fix the cells with 4% paraformaldehyde at room temperature for 15 minutes;

2. Wash the cells twice with PBS for 10 minutes each time, and then permeate the membrane with 0.1% Triton X-100 at 4 ℃ for 15 minutes;

3. Wash the cells twice with PBS for 10 minutes each time, and then block the cells with 4% BSA at room temperature for 30 minutes;

4. Dilute the alpha actin primary antibody in a ratio of 1:100, and then incubate the cells overnight at 4 ℃ in a refrigerator;

5. Wash the cells with PBS three times, each time for 10 minutes. Dilute the secondary antibody against alpha actin in a ratio of 1:150 and place it at 37 ℃ for 1 hour;

6. Wash with PBS three times, each time for 10 minutes, and finally observe the image under an inverted fluorescence microscope and take photos.

Training operation steps:

1. Use cover tweezers to remove the cover glass from 75% ethanol, wipe it clean with a sterile silk cloth, do not use gauze;

2. Gently place the cover glass into a 6-well culture plate (one slide per well) or culture dish (2-3 slides per dish);

3. Expose for 2-3 hours at a distance of 20-30 centimeters from the direct range of the ultraviolet lamp;

4. Transfer the counted cell suspension into a culture plate and immerse the cover glass in the culture medium;

5. Incubate the culture plate in a 5% CO2 water bath at 37 ℃ for 2-3 days. When the adherent cells have grown to cover 2/3 of the bottom area of the culture plate, remove the plate and gently remove the cover glass with tweezers. Rinse with distilled water for rapid fixation and immunohistochemical detection.

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Notes:

1. This kit is for scientific research purposes only and cannot be used for diagnosis or treatment.

2. The reagents in the spiral cap micro reagent tube should be centrifuged briefly before opening the cap, and the liquid on the cap and inner wall of the tube should be centrifuged to the bottom of the tube to avoid reagent loss when opening the cap.

3. Do not mix with other brands of reagents, otherwise it will affect the effectiveness of use.

4. Sample or reagent contamination by bacteria or fungi, or cross contamination of reagents, may lead to incorrect results.

5. It is best to use disposable suction tips, tubes, bottles, or glassware. Reusable glassware must be cleaned and any residual cleaning agents removed before use.

6. Avoid contact between skin or mucous membranes and reagents.

7. Can be stored in the dark at -20 ℃ for a long time. Avoid repeated freeze-thaw cycles, otherwise it will increase blank absorption and affect the test results. It is best to divide in small doses and wrap them in dark bags or black paper or tin foil to avoid light.

8. Prepare the drug to be tested using culture medium or PBS. If the test drug has reducibility, measure the blank absorbance at 490 nm of the test drug solution containing only MTS without cells. If the absorbance is very low, MTS can be directly added. If the absorbance is relatively high, the culture medium needs to be removed and the cells washed twice with fresh culture medium. Then, 100 μ l of new culture medium and 10 μ l of MTS can be added for detection.

9. Cell processing requires careful handling to avoid human damage to cells as much as possible.

10. The length of incubation time after adding MTS solution depends on experimental conditions such as cell type and cell density. For most cases, incubation for 1 hour is sufficient, while white blood cells need to be cultured for a longer period of time.

11. If a 96 well plate is not used for detection, the dosage of MTS solution can be increased proportionally.