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MTT assay kit

NegotiableUpdate on 02/17

Model

Nature of the Manufacturer: Producers

Product Category

Place of Origin

Online Consult

Overview

The MTT assay kit company is currently selling PC-3M (human prostate cancer cells) 5 #215; 106 cells/bottle #215; 2-295 (human XG malignant glioma cells) 5 #215; 106 cells/bottle #215; 2SMC-1 (human pleural tumor cells) 5 #215; 106 cells/bottle #215; 2SW1116 (human colon adenocarcinoma cells) 5 #215; 106 cells/bottle #215; 2-13 (human esophageal cancer cells) 5 #215; 106 cells/bottle #215; two

Product Details

Product Parameters:

Chinese name	English name	Product Specifications	Product Item Number
MTT assay kit	MTT Assay Kit	500 times	BJ-01X6322

MTT is widely used for detecting cell growth, and its principle is that MTT can be reduced by dehydrogenases in the mitochondria of living cells to produce dark purple formazan crystals, while dead cells do not have this activity. After the dissolution of deep purple formazan crystals, their concentration can be determined by measuring the light absorption at a wavelength of 490nm, and the vitality of the cells can be inferred from this. The more vigorous the cell proliferation, the higher the absorbance; The greater the cytotoxicity, the lower the absorbance.

Product Details:

Product Features:
1. Using the Formazan dissolution solution of * can fully dissolve Formazan and reduce errors.
2. Low background, high sensitivity, wide linear range, and good repeatability.
3. This product is sufficient for 500 times (5 96 well cell culture plates) of microplate testing.
4. It can be used for detecting the activity of bioactive factors, screening anti-tumor drugs, conducting cell toxicity tests, and determining tumor radiosensitivity.
Storage conditions: Low temperature transportation, -20 ℃ storage (but solution B can also be transported and stored at room temperature), with a shelf life of one year.
Usage:
The following operation is a test for detecting cell toxicity, and other applications are similar or simpler (such as growth curve testing). The operation steps can be slightly modified based on this, so they will not be repeated here.
1. Inoculate cells
1. Digest the confluent monolayer cells using conventional digestion methods and collect them in a serum containing culture medium.
Centrifuge at 2.200g for 5 minutes to collect cell sediment.
3. Resuspend the cell pellet in culture medium to prepare a single-cell suspension and count.
4. Dilute the cells to a concentration between 2.5 × 103 cells/mL and 5 × 10 cells/mL (depending on the growth rate of the cells). If the growth rate is unknown, it can generally be diluted to 1 × 10 cells/mL.
5. Transfer a sufficient amount of cell suspension into a culture dish (for easy sampling with a pipette). A 96 well MTT assay requires approximately 20mL of cell suspension.
6. Use a pipette to add 200 μ L of cell suspension (for normal cells) to the center of each well in columns 2-11 of the 96 well plate. If it is tumor cells, add 100 μ L of tumor cell suspension and 100 μ L of culture medium (total volume of 200 μ L). Attention: Be sure to place the cells in the center of the well, otherwise the cells will gather in the corners of the well, affecting the experiment.
7. Use a pipette to add an equal volume of culture medium to the cell suspension in the first and twelfth wells of the 96 well plate. The first column of each well will serve as a+medium cell+MTT control (used to zero out during OD measurement), and the addition of medium in the 12th column is to reduce the impact of edge effects on the response in the 11th column.
8. Incubate cells using conventional cell culture methods at 37 ℃ and 5% CO2 for 1-3 days to enter the exponential growth phase.
2. Drug treatment
9. Dilute the drug to 8 test concentrations using culture medium (if the test concentration is unknown, pre testing is required to determine). Generally, one drug requires 3 parallel plates.
10. Remove the culture medium from each well in columns 2 to 11 (out of 10) (without touching the cells), and retain the culture medium from each well in columns 1 and 12.
11. Add 200 μ L of fresh culture medium to each well in columns 2 and 11 (out of 2), which will serve as a control for+culture medium+cell drug.
12. Add 8 concentration gradients of the test drug to each well in columns 3 to 10 (out of a total of 8 columns), with one concentration of drug added to each column.
13. Continue to incubate the 96 well plate under 37 ℃ and 5% CO2 conditions for a certain period of time according to conventional methods. This period of time is the time for drug treatment of cells, which is determined by the user themselves.
14. After processing, remove the culture medium from all wells in columns 2 to 11 (10 columns in total, all containing cells), and add 100 μ L of fresh culture medium.
15. Change the culture daily to increase the number of cells by 2-3 times (the required time varies depending on the cells).
3. Count of surviving cells
16. In the late stage of growth, after removing the culture medium from each well in columns 1 to 11, add 100 μ L of fresh culture medium and 10 μ L of solution A (containing MTT component), wrap the 96 well plate with tin foil, and continue to culture at 37 ℃ and 5% CO2 for 4-8 hours. Attention: Solution A will solidify at low temperatures. Before use, please leave it at room temperature or in a 20-25 ℃ water bath until completely dissolved. Shake well before use. MTT is carcinogenic and must be operated with gloves.
17. Be careful to discard the culture medium (including solution A) in the well. As the culture medium may affect light absorption, it is best to remove it as much as possible.
18. Add 100 μ L of solution B to each well and shake at low speed on a shaker for 10 minutes to fully dissolve the formazan crystals formed by MTT.
Due to the instability of the product, it is necessary to immediately select 490nm for absorbance measurement on an enzyme-linked immunosorbent assay (ELISA) detector.
Attention: Zero each well in column 1 (+culture medium cell+MTT control).
20. Count the average of each repetition processed in the same way.
21. Draw a curve with drug concentration as the horizontal axis and absorbance as the vertical axis. Due to the significant difference in absolute absorbance values among different treatments, it is generally necessary to convert them into growth inhibition rates (using the average of the data from columns 2 and 11 without drugs as 100%), in order to calculate IC50 and compare the treatment effects of various drugs. Note: If measuring the growth curve, use time as the horizontal axis. The normal growth curve generally presents an S-shape, and those with a promoting effect have an increased slope.

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.

Key points and instructions of the experiment:

1. This method is suitable for adherent cell culture, but not for suspension cell culture. Suspension cells can be cultured using the droplet method;

2. The cover glass used should be high-quality glass and treated with chromic acid solution;

3. Cover slips are very thin and fragile, so be gentle when taking and placing them;

If more cells with consistent growth status are needed, larger culture dishes can be used, but they should not be too large to avoid wasting the culture medium and increasing the risk of contamination;

If the cell adhesion growth ability is poor, the cover glass can be soaked in a 0.5% polylysine solution for 5-10 minutes and naturally air dried.

Operation points:

1) Preheat the culture medium in a 37 ℃ water bath; Prepare a 15mL sterile centrifuge tube and add 8mL preheated culture medium.

2) Remove the frozen cells from the liquid nitrogen tank and quickly place them in a 37 ℃ water bath for reheating (a clean beaker can be prepared and filled with 37 ℃ water. After removing the cell freezing tube, quickly place it in the beaker and gradually transfer it to the water bath). Gently shake the cryotube to thaw the cells within 1-2 minutes, allowing them to quickly pass through the most vulnerable temperature range (-5~0 ℃). Be careful not to submerge the freezing tube mouth into water to avoid contamination caused by BRL (rat liver cells).

3) Wipe the cryotube with 75% alcohol and place it in a super clean table. Transfer the cells inside the tube to a prepared centrifuge tube and gently blow the liquid to evenly disperse the cells and reduce the concentration of DMSO. Avoid creating bubbles during blowing. Wash the tube wall twice with fresh culture medium and transfer both to a centrifuge tube.

4) Centrifuge at 800rpm for 5 minutes, discard the supernatant, add fresh culture medium, and prepare a cell suspension by blowing.

5) Transfer the cell suspension to a T25 cell vial, add an appropriate amount of culture medium, gently shake the cell vial to evenly distribute the cells, and place it in a incubator for cultivation.

6) The next day, observe the cell adhesion growth and replace with fresh culture medium to remove dead cells. Continue to cultivate and passage normally when the cells reach 80-90% confluence. Generally, newly revived cells need to be passaged 2-3 times before subsequent experiments can be conducted after the cell vitality is restored.

Angiotensin converting enzyme inhibitor antibody

Antiapoptotic transcription factor antibody

Alpha-1 anti pancreatic

ATP binding protein family 6 antibody

Adenosine triphosphate binding cassette subfamily G1 antibody

Adiponectin receptor 2 antibody

ANGEL1 protein antibody

ANGEL2 protein antibody

ANKLE2 protein antibody

Testicular specific anchor protein 1 antibody

Anchor protein repeat domain protein 13b antibody

Anchor protein repeat domain protein 20A1 antibody

Anchor protein repeat domain protein 20A3 antibody

Anchor protein repeat domain protein 22 antibody

Anchor protein repeat domain protein 50 antibody

BC-020 (human breast cancer cell) 5 × 106 cells/bottle × 2

BC-021 (human breast cancer cell) 5 × 106 cells/bottle × 2

BC-022 (human breast cancer cell) 5 × 106 cells/bottle × 2

BE (2) - M17 (human neuroblastoma cells) 5 × 106 cells/bottle × 2

BGC-803 (human gastric cancer cells) 5 × 106 cells/bottle × 2

C918 (human choroidal melanoma cells) 5 × 106 cells/bottle × 2

CAL-27 (human tongue squamous cell carcinoma cells) 5 × 106 cells/bottle × 2

LS 180 (human colon adenocarcinoma cells) 5 × 106 cells/bottle × 2

CNE-2Z (human nasopharyngeal carcinoma cells) 5 × 106 cells/bottle × 2

MTT assay kitCOLO 201 (human colorectal adenocarcinoma cells) 5 × 106 cells/bottle × 2

CW-2 (human colon cancer cells) 5 × 106 cells/bottle × 2

CRT (human glioma cells) 5 × 106 cells/bottle × 2

D283 Med (human medulloblastoma cells) 5 × 106 cells/bottle × 2

EA.Hy926 (human umbilical vein cell fusion cells) 5 × 106 cells/bottle × 2

ECV-304 (human umbilical vein endothelial cells) 5 × 106 cells/bottle × 2

EJ (human bladder cancer cell) 5 × 106 cells/bottle × 2

H-97 (human highly metastatic liver cancer cells) 5 × 106 cells/bottle × 2