Q1：How to test 24-well plate, 12-well plate in addition to 96-well plate operation example? A1: Add 1:10 ratio with culture medium.
Q2: How to determine the blank well? A2: There are two sources of blank readings, reducing substances and the activity of the cells themselves. Please confirm the following details. -Reducing substances: CCK-8 and the drug to be tested can be added to the culture medium before the formal experiment. If there is a change in color, it proves that the drug has reducing properties. For formal experimental testing, please change the culture medium before adding CCK-8. -Change in cell activity: CCK-8 detects cell activity, so a change in cell activity will change the blank even if the number of cells remains the same. Therefore, it is necessary to confirm whether there is a change in cell activity.
Q3: How to terminate the reaction? A3: There are several methods (96-well plate): 1. After the color development reaction, place the culture plate in the refrigerator at 4℃. 2. Add 10 μl of 0.1 M HCL solution to each well. 3. Add 10 μl of 1% (w/v) SDS (sodium dodecyl sulfate) solution to each well. Note: Measurement should be done within 24 hours after the reaction is stopped. 4. Use our Stop Solution for CCK-8 (Item No. CK13).
Q4: Can I use filters other than 450 nm? A4：Can I use 430-490nm filter, 450nm filter is the best, WST-8 dirty dye absorption spectrum:
Q5: What are the transportation and storage conditions for CCK-8? A5: The reagent can be stored stably at 4℃ for 24 months. If long-term storage is needed it can be stored at -20℃, but do not freeze and thaw repeatedly. If changing to other containers, please conduct stability test first to confirm the reagent stability.
Q6: How many cells should be inoculated in one well? A6: When using a standard 96-well plate, the minimum inoculum of adherent cells is at least 1,000 cells/well (100 μl medium). The sensitivity is relatively low when detecting leukocytes, so an inoculum size of no less than 2,500 cells/well (100 μl medium) is recommended. For experiments using 24-well or 6-well plates, please calculate the corresponding inoculum volume per well and add CCK-8 solution at 10% of the total volume of medium per well.
Q7: Can I use 384-well plate for the experiment? A7: Yes, you can. Add 10% of the medium volume of CCK-8 solution to each well. If the volume of CCK-8 added is too small, you can first dilute CCK-8 solution by 1 times and then add 20% of the medium volume.
Q8：Can we use 24-well plate for the test? A8: Yes, you can. Add 10% of the medium volume of CCK-8 solution to each well.
Q9: Will phenol red affect the test? A9: No. The absorbance of phenol red in the medium can be eliminated by subtracting the absorbance of the background in the blank wells during calculation, so it will not affect the assay.
Q10: Is there a correlation between CCK-8 and thymidine binding assay? A10: Yes. However, please note that since CCK-8 uses a different detection principle than the thymidine assay, the results may be different.
Q11: Can CCK-8 detect bacterial cells? A11: It can detect E.coli, but not yeast cells. Add 10 μl CCK-8 solution to 100 μl E.coli culture medium and incubate for 1-4 hours or overnight. But it is more recommended to use our item number M439: Bacterial Activity Assay Kit.
Q12: How to minimize the error due to the residue of CCK-8 reagent on the tip or well wall? A12：You can dilute CCK-8 reagent with culture medium and mix it before adding sample.
Q13: What color is CCK-8? A13：It should be pink. If the color is different, it may affect the measurement.
Q14: Can CCK-8 stain live cells? A14: It cannot. This is because the main component of CCK-8 is a water-soluble tetrazolium salt (WST®-8) and exchanges electrons from living cells to WST®-8 in the culture medium via the electron carrier 1-Methoxy PMS. Since the generated methoxy PMS is also highly water-soluble, CCK-8 cannot stain the cells.
Q15: Is the CCK-8 assay solution toxic to cells? A15: The CCK-8 solution itself is slightly toxic due to the presence of high concentrations of 1-Methoxy PMS. However, CCK-8 added to the culture medium is not toxic because it is diluted 10-fold. Therefore, prolonged incubation, such as overnight or incubation for several days is possible. The same cell culture can also be used for other cell proliferation assays after the CCK-8 assay, such as the crystal violet assay, neutral red assay or DNA fluorescence assay. Since each cell has a different tolerance for CCK-8, when a long incubation period is required, first test the viability of the cells after adding CCK-8 to the culture.
Q16: When doing the addition experiment, does the drug have any effect on the assay? How to solve it? A16：Sometimes there will be an effect. If the drug is reducing, it will have a color development reaction with CCK-8 and increase the absorbance. Solution: First of all, confirm whether the drug is absorbed or not, add CCK-8 to the medium containing the drug and measure the absorbance at 450 nm. If its absorbance is higher than that of the medium without the drug (with CCK-8), then it proves that the drug has an effect, and the medium can be changed to remove the effect of the drug before adding CCK-8.
Q17：What is the reason that the value is not the same for each measurement? How to solve it? A17：There may be several reasons: 1. When incubated in an incubator, the holes in the outermost circle of the culture plate are most likely to dry out and evaporate, increasing the error due to inaccurate volume. Normally, the outermost circle of wells is only added with culture medium and is not used as assay wells. 2. There is a possibility of error due to CCK8 staining on the walls. It is recommended to gently tap the culture plate after adding CCK-8 to aid in mixing. 3. Too many or too few cells per well. Please feel the conditions in the range of 1,000-100,000 cells/well in advance.
Q18: How do I set up a blank control? A18: Add CCK-8 to the cell-free medium, incubate for a certain period of time, and measure the absorbance at 450 nm as the blank control. When doing the drug addition experiment (cytotoxicity experiment), the absorption of the drug should also be taken into account, and CCK-8 can be added to the medium without cells and with the drug added, incubated for a certain period of time, and the absorbance at 450 nm can be measured as the blank control.
Q19：What substances affect the determination of CCK-8? A19：The determination of CCK-8 is affected by the presence of reducing substances, such as Glucose containing vitamin C. (The amount in the medium is generally small, and phenol red or serum does not affect the determination). In the presence of phenol red, it will increase the blank absorption, but it does not affect the measurement, and the blank absorption can be deducted.
Q20：How to solve the problem if the absorbance value is too high in the experiment and if the number of cells cannot be reduced? A20：You can shorten the incubation time after adding CCK-8. For example, the incubation time after adding CCK-8 reagent can be shortened from 2 hours to 1 hour.
Q21: What is the purpose of setting the reference wavelength? Does it have to be set? A21: It is not necessary to set the reference wavelength; CCK-8 reagent has no absorbance at the reference wavelength. The purpose of setting the reference wavelength is to take out the absorption due to turbidity of the sample.
Q22: The instruction manual only states the measurement method for 96-well plates, if I use a 24-well plate with 12 wells, how much CCK-8 reagent should I add? A22: Generally, it is recommended that the amount of CCK-8 reagent added is 1/10 of the volume of culture medium.
Q23: Do I have to pre-culture the cells? A23: Not necessarily. It is recommended to pre-cultivate the cells if it is necessary toward keeping the cells in the best condition. If you do not do cell pre-culture, the dehydrogenase in the cells may be unstable. Some people also do not do cell pre-culture, but need to standardize the assay conditions when doing standard curves and assays.
Q24: If the drug added contains metal, will it have any effect? A24: Metals have an effect on CCK-8 color development. When the final concentration is 1 Mm, lead chloride, ferric chloride, and copper sulfate will inhibit the color development reaction by 5%, 15%, and 90% and degrade the sensitivity. If the final concentration is 10 Mm, it will inhibit 100%.
Q25: After pre-culturing, do I need to change the medium for cell counting? A25：Generally speaking, it is sufficient to treat the cells in the logarithmic growth phase with trypsin, count them with hemocyte counting disks, and prepare a certain concentration of cell suspension. If you want to count the cells precisely, you can take the culture medium after pre-culture and count them with hemocyte counting disk.
Q26: Does CCK-8 have the same sensitivity for different cells? A26: No. Suspension cells are more difficult to stain compared to adherent cells. For adherent cells, the absorbance is usually already high after adding CCK-8 for 1-4 hours of incubation, but for suspension cells it may be lower, which can be solved by prolonging the addition time of CCK-8 or increasing the number of cells.
Q27: What is the difference in quantity between suspension cells and adherent cells? A27：Suspension cells are more difficult to develop color, so it is generally necessary to increase the number of cells and extend the incubation time. Adherent cells are easier to develop color, if the number of cells is too large, sometimes the absorbance will exceed the reading of the enzyme marker.
Q28: Should I do the standard curve every time? A28: It is recommended to do it every time. Although the cells are the same, the state of the cells is not always the same, for the cells with different states, it is recommended to do the standard curve every time. If the reagents have different lot numbers, there may be a slight difference in sensitivity, and it is recommended to do the standard curve separately for different lot numbers. Q29：Sometimes in the case of drug action, the cells have died, but the dehydrogenase activity is still there, can the number of cells be counted? A29: No. Since CCK-8 indirectly reflects the number of living cells by reacting with intracellular dehydrogenase, it is not possible to calculate the number of living cells.