MTT assay kit

The company's products are for scientific research purposes only. The following are the product attributes:

Product Introduction:
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 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), valid for 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.

Notes:
① The order of adding reagents should be consistent to ensure that the incubation time for all reaction plate wells is the same.
② Use clean plastic containers to prepare detergent.
③ Perform incubation operations according to the time, amount, and sequence indicated in the instructions.
④ Substrate A should evaporate and avoid opening the lid for a long time. Substrate B is sensitive to light and should be avoided from prolonged exposure to light. Avoid contact with hands, toxic. After the experiment is completed, the OD value should be read immediately.
⑤ When washing the enzyme-linked immunosorbent assay (ELISA) plate, it should be thoroughly dried. Do not directly put the absorbent paper into the ELISA reaction well to absorb water.
⑥ Use disposable suction tips to avoid cross contamination, and avoid using sample dispensers with metal parts when suctioning termination solution and substrates A and B.
⑦ The Flat noodles not used in the experiment shall be immediately put back into the packaging bag and sealed for storage to avoid deterioration.
⑧ Reagents should be stored according to the label instructions and returned to room temperature before use. The diluted standard should be discarded and cannot be stored.
⑨ Other unused reagents should be packaged or covered. Do not mix reagents of different batches. Use before shelf life.

Experimental procedures:
(1) Preheat the RNA extraction solution in a 65 ℃ water bath before starting the experiment. Add ME (mercaptoethanol) to a centrifuge tube (add 80ul to 10mL and 300ul to 50mL)
(2) Take about 0.8g of mycelium (liquid cultured mycelium can be filtered under vacuum! Solid cultured mycelium is even better), quickly grind it into fine powder in liquid nitrogen, transfer it into a 50mL centrifuge tube, add 8mL of preheated RNA extraction solution to 1g of material, invert and mix well
(3) 65 ℃ water bath for 3-10 minutes, mix 2-3 times during this period
(4) Add equal volume of phenol (note acidic phenol pH 4.5): Isopentanol (25:24:1) extraction (10000rpm, 4 ℃, 5 min)
(5) Extract the supernatant with equal volume of isoamyl alcohol (24:1) at 10000rpm, 4 ℃, and 5 minutes
(6) Add 1/4V volume of 10M LiCl solution and leave at 4 ℃ for more than 6 hours (or overnight)
(7)10,000rpm, Centrifuge at 4 ℃ for 20 minutes
(8) Discard the supernatant and dissolve the precipitate in 500ul of SSTE
(9) Phenol: Extract twice with isoamyl alcohol (25:24:1), extract once with isoamyl alcohol (24:1) (10000rpm, 4 ℃, 5min)
(10) Add 2V volume of anhydrous ethanol and precipitate at -70 ℃ for more than 30 minutes in a refrigerator
(11)12,000rpm, Centrifuge at 4 ℃ for 20 minutes
(12) Discard the supernatant Rinse the precipitate once with 70% alcohol and dry it
(13) Dissolve 200ul of DEPC in treated water
(14) Detection of RNA quality by non denaturing agarose gel electrophoresis and UV spectrophotometer scanning
(During the extraction process, if the protein content or other impurities are still high, the number of extractions can be increased)

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Operating procedure:

Attention: The most important thing is to fix it in a timely manner and add 0.1% DEPC treatment to the fixative to inhibit the degradation of mRNA by RNAse. In addition, excessive fixation has a significant adverse effect on in situ hybridization.

1. Processing of glass slides: Polylysine or APES are generally used. Slice thickness 10-20 μ m.

2. Cells were cultured on cover slips treated with polylysine under conditions of 37 ℃ and 5% CO2, using Dulbecco basic medium. After the cells have grown well, wash them with 0.1M PBS (pH 7.4) for 2 minutes x 3 times.

3. Both cultured cells and frozen sections can be fixed using the following method: the fixative is 4% paraformaldehyde/0.1 M PBS (pH 7.2-7.6) containing 1/1000 DEPC. Fix at room temperature for 20-30 minutes. Thoroughly wash with distilled water, dry and freeze at -20 ℃ for more than 2 weeks.

Mix 1 part of 4.30% H2O2 and 50 parts of pure methanol, and treat at room temperature for 30 minutes. Wash with distilled water three times.

5. Exposure of mRNA nucleic acid fragments: Add 1ml of freshly diluted 3% citric acid and 2 drops of concentrated 3% citric acid to the slice, mix well, and digest at 37 ℃ or room temperature for 5-120 seconds. Sometimes it can also be undigested. Wash in situ hybridization with PBS 3 times for 5 minutes. Distilled water wash once.

6. Post fixation: It is only necessary after digestion. The fixative is 1% paraformaldehyde/0.1 M PBS (pH 7.2-7.6) containing 1/1000 DEPC. Fix at room temperature for 10 minutes. Wash with distilled water three times.

7. Pre hybridization: Preparation of wet box - Add 20ml of 20% glycerol to the bottom of the dry hybridization box to maintain humidity. Add 20 μ l of pre hybridization solution to each slice. Thermostatic chamber at 38-42 ℃ for 2-4 hours. Absorb excess liquid without washing.

8. Hybridization - Add 20 μ l of hybridization solution to each slice. After removing the protective film of the in situ hybridization special cover glass, cover it on the slice. Hybridization overnight at 38-42 ℃ in a constant temperature incubator (adjustable according to hybridization conditions).

9. Wash after hybridization: Remove the cover glass and wash with 2 times SSC at a water temperature of around 37 ℃ for 5 minutes twice; Wash with SSC at 37 ℃ for 15 minutes once; Wash at 37 ℃ for 15 minutes with 0.2 times SSC (if there is non-specific staining, repeat 0.2 times SSC washing for 15 minutes with 1-2 times).

10. Add blocking solution dropwise: 37 ℃ for 30 minutes. Discard excess liquid without washing

11. Add Hua Shu Kang * dropwise at 37 ℃ for 60 minutes or at room temperature for 120 minutes. Wash in situ hybridization with PBS for 5 minutes x 4 times. Do not wash with other buffer solutions or distilled water.

12. Add SABC dropwise at 37 ℃ for 20 minutes or at room temperature for 30 minutes. Wash in situ hybridization with PBS for 5 minutes x 3 times. Do not wash with other buffer solutions or distilled water.

13. Dropwise addition of peroxidase: at 37 ℃ for 20 minutes or at room temperature for 30 minutes. Wash in situ hybridization with PBS for 5 minutes x 4 times.

14. DAB color development: Use DAB color development kit -1ml distilled water with one drop each of color development agents A, B, and C, mix well, and add to the specimen. Generally, the color development takes 20-30 minutes. If no background appears, the color can continue to appear. It can also be developed with self prepared DAB color developer. Thoroughly wash with water.

15. If necessary, counterstain with hematoxylin and wash thoroughly with water.

16. Dehydration with alcohol, transparent with xylene, and sealed.