Keeping cells, pay attention to these details!(1)
Keeping a cell is one of those things that poke your heart out and you have to treat it like a child, love her and care for her.
If you pay attention to these problems when you're taking care of them, you'll make your cells better.
Next, we will talk about the care of the cell.
Preparation before cell culture
Before you bring gloves start to cell culture, to check the pipet and adequacy of the number of bottle, lest you start again after the experiment in and out of work station, so that we can reduce the risk of pollution of cells.
Cell culture medium should also preheat, first choose only preheat medium rather than the whole bottle of heating, not only can save the time, but also can avoid the protein degradation caused by repeated heating medium.
After finishing the operation, don't forget that the medium is sensitive to light and should be kept as light as possible.
Regular examination of cell culture
Regular examination of the formation of cells, that is, shape and appearance, is critical to the success of cell culture experiments.
In addition to confirming the healthy state of the cell, the cell can be detected by the naked eye and microscopic examination of the cell during each operation.
A sign of cell metamorphism
The image of cell metamorphism includes the occurrence of particles, cell and matrix dissociation and cytoplasmic vacuole formation.
These metamorphic signs may be caused by a variety of causes, such as:
There are toxic substances in culture, cell lines, aging or medium, or these signs only indicate that the culture medium is needed.
When it goes bad, it's going to be an irreversible change.
The sterilization and layout of the cell culture fume hood
Keep the cells in a clean and orderly room and keep all items in the open air.
Spray 70% ethanol to all items in the fume hood and wipe clean for disinfection.
Place cell culture containers in open Spaces in the middle of the fume hood;
The liquid transfer device is easy to be used in front of the right.
The reagent and culture medium were placed on the right and convenient for absorption.
The test tube holder is placed in the middle;
Small containers are placed at the back of the left and used for holding waste.
To cultivate the sterile operation of bottles/dishes and other items
Sterile culture bottle, reagent bottles, the article such as dish can uncover the lid when using, must not be exposed its open environment, the operation as soon as possible after the completion of a lid on it, when take off the lid, should open the lid down on the table.
Do not speak, sing or whistle while doing aseptic operation.
Complete the experiment as soon as possible to avoid contamination.
Possible contaminants in cell culture
There are two main types of cell culture pollutants:
Chemical contaminants such as impurities, endotoxins, plasticizers and detergents in media, serum and water.
The cross-contamination of biological pollutants such as bacteria, molds, yeast, viruses, mycoplasma and other cell lines.
The frequency and severity of pollution can be reduced by fully understanding pollution sources and adopting good aseptic technology.
Cross-contamination confirmation
Cross-contamination is not as common as microbial contamination, but widespread cross-contamination with HELA cells and other rapidly growing cell lines is a clear problem that can have serious consequences.
Obtaining cell lines from reputable cell libraries, checking cell lines regularly and adopting good sterile techniques can help avoid cross-contamination.
DNA fingerprinting, karyotype analysis and isotope analysis can confirm the presence of cross-contamination.
The cell exchange notes
When replacing the fluid, gently add it along the side of the culture bottle instead of directly into the cell, so as not to damage the cell, especially when the cultured cell lines are more vulnerable.
When using a sterile glass straw or a disposable plastic straw and a pipette to operate the liquid, each straw can only be used once to avoid cross-contamination.
The time of cell transmission
Exponentially when the cell proliferation, adherent cultured cells has room for all the available substrate, no amplification, or suspension culture cells already exceeds the ability of the development the substrate support, failing to further growth, cell proliferation rate will be greatly reduced or even stopped completely.
In order to maintain the cell density at optimum levels so that cells continue to grow and stimulate further proliferation, the cells must be passed on.
The use of antibiotics in cell culture
In cell culture, antibiotics should not be used for long periods of time, because continuous use of antibiotics can promote the production of antibiotic resistant cell lines, resulting in a persistent mild pollution.
Antibiotics can only be used as a last resort against pollution and removed as soon as possible.
If antibiotics are used for a long time, they should be cultured at the same time without antibiotics in order to be used as a control for the identification of recessive infection.
The need for cell freezing
Continuous culture cell lines are prone to genetic drift, finite cell line will eventually happen aging, all cultured cells are susceptible to microbial contamination, even the best of running laboratory equipment failure problems.
Since the established cell lines are a valuable resource, replacement of cell lines is costly and time-consuming, so it must be frozen and stored for a long time.
The matter of cell freezing
Cell culture freeze should be carried out in high cell concentration, and the number of cell transmission should be as few as possible.
Ensure that the percentage of living cells is at least 90% before freezing.
Please note that the best frozen conditions depend on the cell lines used.
Frozen storage and recovery process of most cells can cause adverse effect, so do not force by the vortex shock or knock culture flask method makes the cell falls off (with the exception of cultivation of insect cells), and also don't high-speed centrifugal cells.
So be familiar with it, and board it as soon as possible.
prev:Keeping cells, pay attention to these details!(2)(2017-09-13)
next:Trypsin digestion for subculture of adherent cells(2017-09-14)