Cell culture plates are commonly used and important consumables in cell culture experiments because they can save time and reagent materials for cell experiments, and can set up multiple dynamic variables at the same time to facilitate observation and detection.
culture plate
1. The lids of 96, 24-well culture plates or petri dishes are very loose, which is convenient for ventilation, but will bacteria, molds and other pollutants also slip in?
Answer: (1) The cover is very loose and belongs to semi-open culture. The purpose of this is to breathe (actually, to enable the CO2 outside the culture dish to be fully exchanged with the culture dish to maintain the pH value of the medium).
(2) There are advantages and disadvantages, which increases the possibility of pollution. In addition, this causes the liquid in the petri dish to evaporate, which is notable for precise dosing of drugs. So the following two measures are necessary:
a) The air in the incubator must be clean (regular ultraviolet light, alcohol scrubbing, and the incubator should be switched on and off as little as possible)
b) Humidity in the incubator must be maintained at 100% at all times (a sink in which sterile distilled water is placed in the incubator). Pay attention to aseptic operation!
2. My cells are inoculated on the culture plate, and the cells always gather in the peripheral part. What should I do? I see that the cells of some comrades in arms are gathered in the middle. Is it a problem with the quality of the plate?
A: How do you mix your cells? Is it pipette blowing or shaking the culture plate? If it is the latter, and it is shaken in a circle, it is very likely that the cells will be thrown to the surrounding part due to the centrifugal force, resulting in the middle of the cells. Less, more than four weeks! You can try it yourself!
A good way: before you cultivate the seed plate, put the culture plate into the incubator for a few hours of saturation and then take it out. When seeding the cells, the force should be light. You can use a dripper on the side of the culture plate hole slowly Add to allow the cell suspension to flow into the wells of the plate, and the cultured cells grow substantially uniformly. Remember to never shake the oscillator or your cells will clump together like you said.
3. My cells are passaged normally, but when one type goes into a six-well plate (regardless of drug addition and control), there are always two or three wells (random) cells that do not grow well, are small, and seem to be broken. Has anyone encountered this? What is the reason?
A: It may be related to the temperature of the liquid you add. If your cells have just been taken out to change the medium and add the medium, and the medium is also taken out from the 4 degree refrigerator soon, it is easy for the cells to freeze to death. It is recommended that you best incubate the medium in the incubator for 15min first.
In addition, it is also related to the growth state of your cells. Cells prior to dosing can be incubated with high serum concentrations. Make sure the cells are in good condition.
Or it can be incubated in a 37-degree water bath, or the problem of the culture plate itself, you can try another culture plate. Another possibility is the cell state problem, adjust the serum concentration when seeding the plate.
4. In recent days, I used a six-well culture plate to inoculate cells. After culturing for 24 hours, I changed the medium. Before changing the medium, I observed that the cells adhered to the wall, and the state was very good. After changing the medium, I used the microscope immediately. It was observed that almost half of the cells in each well showed a near-death state. The cells become very small and produce a lot of debris, while the other half of the cells are normal, there is a watershed between abnormal cells and normal cells, the upper half circle is good, the lower half circle is not good, how is this? What's the matter?
A: You can see if there are any of the following reasons
a. The culture plate is not placed horizontally;
b. How about your culture medium, if the culture medium expires, the cells will not adhere. Try a freshly prepared medium
c. It may be the problem of the plate. It is no problem to change the plate of another brand or use the culture bottle.
d. Did you pay attention to the influence of the fan when changing the medium, especially when there are many culture plates that need to be replaced, it is easy to cause the cells to shrink and rupture due to wind and water loss. If so, the medium should be changed one by one, and don't be afraid to waste the pipette, pay attention to the efficiency of the operation!
5. It has been four days since the cells were digested and inoculated into a 24-well plate, and the middle part of each well was always full. The medium was washed with PBS every day, and the same situation occurred when the medium was changed the next day. The edges are well long and the center is full of dead cells?
Answer: If the cells are dense around and sparse in the middle, there are approximately the following reasons:
The medium was added too little. Mainly due to the problem of liquid tension.
Whether there is less water at the bottom of the incubator;
The shaking after cell seeding was too much. The cells are distributed around due to centrifugal force.
The key to whether the cells are evenly distributed is that each type of cell has individual differences, and other people's cell operations may not be suitable for you. So you have to explore on your own and find a set of rules that are unique to your own cells. You have the following experience:
(1) All cell suspensions to be seeded must be mixed with a pipette before seeding.
(2) According to the records, the liquid volume of the 24-well plate is 1ml, in fact, the volume of 1ml is enough.
(3) When inoculating, add the sample slowly, and remember to rotate the pipette tip slightly. The sample can not be added too fast to avoid cells from accumulating at one sample addition point, and pay attention to adding samples in different parts, that is, while adding the movable gun. Head, artificially make the cells tend to be evenly distributed. Doing so has the effect of evenly distributing the cells.
