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Cell Metabolism Assay Experiment Frequently Asked Questions

Time：

2023-05-16

The culture medium was aspirated and the cells were gently washed over with cold PBS (0.01 M, pH 7.4), then the cells were collected by trypsin digestion or cell scraping, 2-5 mL of PBS (0.01 M, pH 7.4) was added to make a cell suspension, and the cells were collected by centrifugation at 4°C, 1000 × g for 5 min.

　　1 How are walled cells collected?

　　Aspirate the culture medium, wash the cells gently with cold PBS (0.01 M, pH 7.4), then collect the cells by trypsin digestion or cell scraping, add 2-5 mL of PBS (0.01 M, pH 7.4), make a cell suspension, centrifuge at 4°C, 1000×g for 5 minutes and collect the cells.

　　2 Can I use trypsin for digestion when collecting walled cells?

　　You can use trypsin to digest the cells, or you can scrape off the cells with a cell scraper.

　　3 How to perform cell counting

　　The common methods of cell counting are blood count plates, flow cytometers or automated cell counters/meters, which can be selected according to the equipment in the laboratory

　　4 How to store the cell sample if it is not measured temporarily?

　　If the cell sample is not measured temporarily, it can be stored at low temperature to avoid repeated freezing and thawing. Generally -20℃ can be stored for half a month, -80℃ can be stored for 1 month. Except for special indexes with higher requirements on the sample. Prepared cell homogenates are best measured on the same day.

　　5 What is the meaning of cell sample homogenization? What are the ways of homogenization?

　　Homogenization means that the cells are homogenized by adding a specific solution to the sample and then homogenizing it in some way. The common ways of homogenizing cells are as follows:

　　a. Manual homogenization: collect a certain number of cells, add homogenization medium, hold the homogenization tube in the left hand and put the lower end in the ice bath, insert the pounding rod vertically into the casing with the right hand, turn up and down and grind dozens of times (5-8 min) to make the cells fully homogenized; or pour it into the mortar, add liquid nitrogen to grind, grind fully, add homogenization medium, and then aspirate the prepared homogenization solution into the EP tube for backup.

　　b. Mechanical homogenization: The samples were loaded into EP tubes, homogenizing medium was added, and cell homogenization was made by using a homogenizer at low temperature, 60 Hz, 90 s.

　　c. Ultrasonic crushing: Cells were crushed by ultrasonic treatment with an ultrasonic generator at an amplitude of 14 μm for 30 s; or by ultrasonic cell crusher, 200 W, 2 s/time, with a gap of 3 s and a total time of 5 min.

　　d. Repeated freeze-thaw: resuspend the cells with homogenization medium, and then subject the cell suspension to a "freeze-thaw-freeze" cycle as follows: place them at -80°C for 1 hr/liquid nitrogen for 0.5 hr, and then place them in a 30°C water bath with slight shaking to make them thaw rapidly, and repeat about 3 times. This method can compromise the viability of some enzymes, so repeated freeze-thawing to fragment cells is not recommended when testing cell samples for enzyme viability.

　　6 Can cell samples be treated with lysate?

　　Since the basic principle of metabolic assay kits is based on chemical reactions, and the components in lysate are relatively complex, there may be components that interfere with the reactions of the kits, so it is not recommended to use your own lysate to treat the samples. Elabscience has developed kit-specific lysates (E-BC-L001, E-BC- L002), which can be used with the kit.

　　7 Do I need to dilute the sample for cell sample testing?

　　Generally, for most of the indicators, cell samples do not need to be diluted. However, it is recommended that 2-3 samples be selected for pre-testing prior to the formal assay to determine if dilution is required and the appropriate dilution level, taking into account sample variation, modeling and other factors.

　　8 Why is it necessary to measure the protein concentration of the sample when measuring cell samples for some indicators?

　　In general, when measuring cell samples, the homogenization process should be carried out first, and the protein released from the homogenized samples is different, so the protein measurement is to correct the degree of homogenization.

　　9 Why is the color of the sample light or almost non-existent when measuring cell samples?

　　It is possible that the content of the indicator in the cell sample is low and the color cannot be observed by the naked eye, but it can be detected by the instrument.

　　10 What should I do if the measured value of the cell sample is very low and not much different from the blank?

　　It is possible that the content of the indicator in the cells is low, you can try to increase the cell concentration, increase the amount of cells or reduce the amount of homogenization medium added (need to exceed the minimum volume of the upper sample), if you have questions, you can consult technical support.