Selection and use of cell culture plates
Selection and use of cell culture plates
The principle of strict sterility in performing cell operations also applies to cell culture plates. All operations must be standardized and scientific, with no additional effects on cell growth. One of the most common problems is to ensure uniformity of the cells after addition of the sample and to minimize the effects of medium change on the growth state of the cells.
Ask:
96-well cell culture plates and 24-well cell culture plates or Petri dishes are very loose, which is convenient for aeration, but will bacteria, molds, and other contaminants slip in?
Answer:
The lid is very loose and is part of a semi-open culture. This is for aeration (actually so that the CO2 outside the Petri dish can be fully exchanged with the Petri dish to maintain the pH of the medium).
Everything has advantages and disadvantages, which of course increases the possibility of pollution. In addition, this causes the liquid in the petri dish to evaporate, which is notable for the precise dosing of drugs. Therefore, the following two measures are necessary a. The air in the incubator must be clean (regular UV light, alcohol scrubbing, and switching on and off the incubator as little as possible) b. The humidity in the incubator must always be kept at 100% (a water tank with sterile distilled water is placed in the incubator ).
Just like a petri dish, it is also a container with an upside-down lid and will not contaminate. The main reason is that the "L"-shaped edge of the cover generates negative air pressure so that microorganisms are attached to the dust, and the dust carried by the airflow cannot pass through the edge of the cover that generates the negative pressure. The ventilation effect is only through the diffusion of air, and no airflow will be generated, so it will only breathe and not penetrate bacteria.
Ask:
Using a 24-well plate, in some of the wells, there are manipulations (inside the bench), while other wells still have cells to grow. I'm worried about contamination this way, don't know what to watch out for.
Answer:
In the ultra-clean bench, if the operation is standardized, it should be possible. I think you can make full use of the cover of the culture plate, try to expose only the holes to be operated, and cover other holes with covers.
Consider it comprehensively before use, and make full use of all the holes; if you only need to use a few holes, you can use only one side, and cover the rest with a lid. I am used to using the right hole first (the right hand is convenient for adding samples).
When operating, use a few glass slides to raise one side of the board, do not open the lid fully, generally no problem.
Uneven distribution of cells and solutions.
Ask:
When cells are seeded on a culture plate, cells always gather in the peripheral part, what should I do?
Answer:
How are your cells mixed? Is it pipetting or shaking the plate? If it is the latter, and if it is shaken in a circle, it is very likely that due to the centrifugal force, the cells are thrown to the surrounding part, resulting in fewer cells in the middle and more around!
Here is a good way: before culturing 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. Add slowly 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 as you said.
The smaller the hole diameter of the culture plate, the more obvious this phenomenon is. The phenomenon of 24- and 96-well plates is unavoidable because the wall of the liquid makes the culture medium in the hole not a liquid level, but the periphery is high, just like a concave mirror, However, due to the need for intervention and other reasons, the use of these two kinds of well plates cannot add enough culture medium, so that the cells appear "edge aggregation" together with the culture medium. Depending on what kind of indicators you need to observe, if it is MTT, immunohistochemistry will affect the results due to cell layering.
When digesting cells, pay attention to pipetting evenly to avoid cell clumping, and the amount of culture medium in the wells should be sufficient. Generally, a sufficient amount of culture medium should be added during inoculation, and the medium should be changed once when intervention is added. , in this case, the phenomenon of "edge set" will be improved, so give it a try.
Ask:
What I am doing is the plaque formation experiment. It is best to spread the cells in a uniform layer, but most of the holes are fine when inoculating the virus, and some are not uniform. There are great differences within the cell group. I would like to ask you what methods you have when adding cells.
Answer:
After cell digestion, pipetting into a single-cell suspension is critical! Make sure the number of cells in each well is the same when splitting!
Of course, the parallelism between the wells of the processing factor is also very important. For example, when adding medicine, the medicine should be diluted and mixed to ensure that the concentration of each well is consistent!
In addition, when adding cells and drugs, the test group and the control group should be added in turn to avoid the difference in cell density and drug concentration caused by the sequence of sample addition!
Ask:
The blowing is already uniform, but the plank, 6 holes, and 24 holes are always a little uneven, and they are a little concentrated in the middle. And it is clear that the counting is done, and the density of each hole is different after a day or two, which will affect the detection. Is there a good strategy?
Answer:
If you use a Transfer pipette, do not suck too much each time, because the cells in the pipette will sink automatically, and there are often many cells in the first few wells, and the cell suspension is often uniform, and the liquid is added to go S-shaped. route.
If a pipette is used, the tips should be handled, and the tips should be removed to avoid damaging the cells so that each pipette corresponds to one well. It is more accurate to add one-to-one holes with a tip. But also pay attention to mixing the suspension.To set up a parallel-group, n should be large enough (it can be calculated).Do not shake after plating as shaking can cause cells to aggregate towards the middle. It's best to do it all at once.
