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

Q：CCK-8 test solution toxic to cells?

The CCK-8 solution itself is slightly toxic due to the presence of high concentrations of PMS. However, CCK-8 added to the medium was not toxic because it was diluted 10-fold. Therefore, prolonged incubation, such as overnight or for several days, is acceptable. The same cell culture medium after CCK -8 detection can also be used for other cell proliferation detection, such as crystal violet detection, neutral red detection or DNA fluorescence detection. Since each cell has a different tolerance to CCK-8, the viability of the cells after addition of CCK-8 in culture is first examined when a long period of culture is required.
Q：Is there a difference between skim milk and BSA seal?

In general, BSA produces clearer results because it has fewer proteins and antibodies have less chance of cross-reacting with it. However, this is not always the case. Some antibodies work better in skim milk, which contains more types of blocking proteins and can block more different types of proteins.
Q：How many cells do WB experiments typically require?

5 × 106 is usually sufficient. But depending on which cell it is, some cells are small and some are spread out. It takes 105 cells to perform routine WB on a single pore. But it is also related to the target protein and the method of extraction. If it is a membrane protein or an organelle distribution of protein cells have increased.
Q：Can plant cells use the liji biological mitochondrial marker kit?

Can mark plant cells, but because plant cells have cell walls, will block the dye into the cell, the need for customers to make protoplasts for staining
Q：Can Annexin V kit detect apoptosis in cells other than human?

Yes, because Annexin V binds to phosphatidylserine (PS) , and there is almost no interspecies difference in PS.
Q：What should be noted for efficient cell cryopreservation?

1) slow freezing and freezing can dehydrate the cells gradually, and the cells will not be damaged by the formation of large ice crystals. On the contrary, the formation of large crystals in cells may cause damage and rupture of cell membranes and organelles. 2) the viability and concentration of cells should be cryopreserved when the density is about 80-90% and the viability is more than 90% . The survival rate of cells with poor viability after cryopreservation is very low. Therefore, it is necessary to cryopreservation during the period of vigorous cell division. 3) cryopreservation density when the cell density is less than 5x105 cells/mL, it is difficult to revive successfully. 4) frozen storage tube cover frozen storage tube cover must be tightened, otherwise the water bath recovery will cause water infiltration pollution. 5) the cells could not be stored in -80 ° C refrigerator for a long time.
Q：When is the best time for cell cryopreservation and resuscitation?

There are three stages of cell growth in vitro: latency, exponential growth and plateau. The incubation period is usually just when the cells begin to attach to the matrix and stretch the cytoskeleton. The metabolic activity focuses on the expression of adhesion and cytoskeleton proteins, and the cell quantity hardly increases during this period. After that, the cells began to grow exponentially, the transcription and translation of nucleic acids were vigorous, the DNA was unrotated and paired frequently, the material in the cells transferred rapidly, and the number of cells increased rapidly. If cryopreservation at this time, in effect, forces the cell to freeze at some point in its metabolism, it is bound to cause mechanical damage to unrotated DNA, RNA and proteins in transcription and translation, increase the risk of errors in individual links. With the increase of the number of cells, the confluence of cells reached more than 90% in the culture container. Most of the cells stopped high metabolism and proliferation because of“Contact inhibition”, and the intracellular activity tended to be smooth. Therefore, we propose to freeze the cells just into the plateau period, both to obtain sufficient cells, but not to cause excessive mechanical damage to the cells.
Q：What is the efficiency of transfection reagent to single plasmid and multi-plasmid co-transfection?

The efficiency of single transfection is very good for the efficiency of verified cells, which can be referred to the verified cell lines, so specific efficiencies need to be verified.
Q：Can transfection reagents be frozen?

Can not be cryopreserved, because the transfection reagent is a cationic polymer transfection reagent, because the polymer is not able to be cryopreserved at low temperature, therefore the transfection reagent is best stored at 4 ° C to maintain the best transfection efficiency.
Q：Does the transfection reagent need to be changed after transfection?

For the change of fluid, we can distinguish two cases: 1. If there is no change of fluid before transfection, we should change the fluid 6-8 hours after transfection to ensure the growth of cells, 2. If there is change of fluid before transfection, 2, you can change the liquid as usual until the nutrient deficiency appears in the medium. The transfection reagent itself is not toxic.

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