Frequently Asked Questions - Answers to Reagent Product Issues-He Yuan Li Ji

Q：Why is the repeatability of CCK8 detection data poor?

1. The reagents in the outermost circle of the well plate are prone to volatilization. It is recommended to only add culture medium to the outermost circle of the well and not use it as a measuring well. 2. CCK-8 sticks to the wall. It is recommended to add CCK-8 and gently tap the culture plate to help mix. 3. If there are too many or too few cells, it is recommended to explore the conditions within the range of 1000-100000 cells per well in advance. 4. Bubbles inside the well interfere with the enzyme-linked immunosorbent assay (ELISA) detection. Before detection, it is necessary to ensure that there are no bubbles in each well.
Q：Why is the absorbance value measured by the CCK8 kit low?

1. The number of cells is too low. The number of cells to be tested can be appropriately increased. 2. Staining is difficult, it is recommended to increase the incubation time of CCK-8.
Q：What is the formula for calculating the survival rate of CCK8 cells?

Vitality Calculation: Cell Vitality × (%)=[A (Dosing) - A (Blank)]/[A (0 Dosing) - A (Blank)] × 100 A (Dosing): Absorbance of pores with cells, CCK-8 solution, and drug solution A (Blank): Absorbance of pores with culture medium and CCK-8 solution but no cells A (0 Dosing): Absorbance of pores with cells, CCK-8 solution but no drug solution
Q：How long can cells be stored in liquid nitrogen?

Cell cryopreservation can temporarily remove cells from their growth state and preserve their cellular characteristics. Cells are stored in liquid nitrogen at a temperature of -196 ℃, and theoretically, the storage time is infinite.
Q：When is the best time for cell cryopreservation?

During in vitro growth, cells are usually divided into three stages: latent period, exponential growth period, and plateau period. The incubation period usually occurs when cells have just been passaged, at which point they begin to attach to the matrix and extend the skeleton. Metabolic activity is focused on the expression of adhesion proteins and skeleton proteins, and the cell count hardly increases during this period. Subsequently, the cells begin an exponential growth phase, with vigorous nucleic acid transcription and translation, frequent DNA unwinding and pairing, rapid intracellular material transfer, and a rapid increase in cell numbers. If frozen during this period, it is actually forcibly freezing the cells at a certain moment of metabolism, which will inevitably cause mechanical damage to the untwisted DNA, RNA and proteins involved in transcription and translation, and increase the risk of errors in individual processes. As the number of cells increases, the confluence of cells in the culture container reaches over 90%. Most cells stop high-speed metabolism and proliferation due to "contact inhibition", and intracellular activity tends to flatten out. So, we suggest freezing cells at the beginning of the plateau phase, which can obtain sufficient cells without causing excessive mechanical damage to the cells.
Q：What should be noted for efficient cell cryopreservation?

1. Slow freezing: Slow freezing can gradually dehydrate cells, and cells will not be damaged by the production of large ice crystals. On the contrary, the appearance of large crystals inside cells can cause damage and rupture of cell membranes and organelles. 2. Cell viability and concentration: Cells should be frozen in a state of good growth, density of about 80-90%, and viability of over 90%. Cells with poor cell viability have a low survival rate after cryopreservation, therefore, they must be cryopreserved during the period of vigorous cell division. 3. Freezing density: When the cell density is less than 5x105 live cells/mL during freezing, it is difficult to successfully recover. 4. Cryovial cap: The cap of the cryovial must be tightened, otherwise water will seep in and cause contamination during water bath recovery. 5. Freezing time: Cells cannot be stored in a -80 ℃ freezer for a long time. We suggest that the storage time in a -80 ℃ refrigerator should not exceed 48 hours.
Q：Why do protective agents need to be added to cell cryopreservation?

Cell cryopreservation is a technique that places cells in a low-temperature environment to reduce cellular metabolism for long-term storage. Cells are frozen without any protective agents, and the water inside and outside the cells quickly forms ice crystals, causing a series of adverse reactions. Firstly, some proteins denature due to increased local electrolyte concentration and pH changes, leading to changes in the internal spatial structure of the cells. Secondly, the formation of ice crystals within cells and the denaturation of proteins and enzymes on the cell membrane system can limit the energy metabolism of cells. Once again, the lipid protein complexes on the cell membrane are prone to damage during freezing, leading to changes in membrane permeability and loss of cellular contents. Finally, as the freezing temperature decreases, the volume expansion of ice crystals causes damage to the spatial structure of DNA, leading to cell death. Adding cryoprotectants can effectively improve cell damage and death.
Q：The difference between PBS solution and DPBS solution

PBS can provide a relatively stable ion environment and pH buffering capacity, and is generally used as a solvent to dissolve and protect reagents. It is a commonly used buffer salt solution in biology, used for molecular cloning, etc. DPBS is a balanced salt solution used to maintain cell activity in the short term. Maintain the structural and physiological integrity of ex vivo cells within a limited time. The pH is 7.2-7.4, which is similar to the pH of the human body. The solution has an osmotic pressure equivalent to that of human blood and does not contain Ca2+or Mg2+. It is suitable for various cell culture applications, such as washing cells before dissociation, transporting cells or tissues, diluting cells for counting, and preparing reagents. In fact, PBS is a general term for a type of phosphate buffered saline solution, although there are commonly used formulas, it is not limited to any one. DPBS is a PBS solution that Renato Dulbecco improved based on his own experimental needs. Due to its frequent use, it has been adopted as a relatively fixed name. Essentially, both belong to phosphate buffer solutions. According to commonly used formulas, the components of the two solutions are basically the same, with slightly different contents. It can be said that DPBS is a type of PBS.
Q：How much is the 1640 culture medium stored at?

In the laboratory, the culture medium should be stored in a refrigerator at 4 ℃. Generally, it is not recommended to store the culture medium at -20 ℃ because the salts in the culture medium are prone to precipitation at -20 ℃. After the culture medium melts, some salts may not be able to dissolve again, resulting in a decrease in the osmotic pressure of the culture medium. When cultivating cells, they are prone to rupture and die. During the storage process, it is also necessary to avoid exposing the culture medium to high temperature environments. Before use, the culture medium needs to be restored to room temperature before use.
Q：What supplements can be added to RPMI1640 culture medium?

We usually add some supplements to RPMI1640 medium, such as growth factors, proteins, 10% fetal bovine serum, phenol red, bispecific antibodies, etc. These supplements are adjusted appropriately according to the type of cells being cultured. For example, if we want to observe the pH value of RPMI1640 medium more intuitively, we can add phenol red. Phenol red produces different colors according to changes in pH value, which is beneficial for researchers to adjust pH value in a timely manner. This is also one of the reasons why RPMI1640 medium changes color; When we want to prevent cell contamination, we can add a dual antibody solution appropriately for antibacterial purposes; We need abundant growth factors and nutrients, so we can simply add 10% fetal bovine serum to ensure that the nutrients in RPMI1640 medium are appropriate. The addition of supplements to RPMI1640 medium is based on our experimental design and objectives.

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