I. Selection of foreign body samples and matters needing attention
Different experimental samples need to pay attention to different points when collecting. What kind of samples are you studying and what aspects do you need to pay special attention to? Look at the following explanation:
1, cell supernatant
Choosing cell culture supernatant as the experimental material for foreign body separation will not have the problem of sample size limitation like blood samples, and it is simple to operate and can be directly centrifuged. However, attention should be paid to pollution, culture time, cell mortality and FBS during cell culture. Like all cell experiments, pollution is not allowed in the culture process, but some cultured cells carry virus genomes themselves, and virus particles will be released during the culture process, which depends on whether the virus particles will affect the subsequent experiments.
Generally speaking, foreign bodies can be isolated from cells after 24-48h culture. Under special circumstances, if special chemicals are added to the culture medium, it can be collected after 15-60min. Because cell death secretes corpuscles, the cell death rate must be controlled within 5% when studying foreign bodies. The untreated FBS contains a large number of foreign bodies, so it is necessary to use the treated FBS without foreign bodies for cell culture.
2. Saliva
The composition of saliva will change with the location and time of sampling, so it needs to be fixed and timed like a blood sample. Saliva is mainly secreted by sublingual gland, submandibular gland and parotid gland, so the composition of saliva in different positions is different, so it is necessary to fix the position or collect all saliva when collecting. The composition of saliva will be different at different time periods, so it needs to be taken regularly. Others, such as smoking and eating, are forbidden. Generally speaking, it is recommended not to eat anything, including drinks, or do strenuous exercise during sampling 1h, and to sample in a calm state. Finally, don't brush your teeth.
3. Blood
In the first exocrine course, many students raised questions about the choice of serum and plasma. Which one should we choose? Plasma = blood-blood cell serum = plasma-fibrinogen-coagulation factor
If you can't tell the difference between the two, you can remember it firmly by finding out the source of serum. Serum is the liquid collected after blood coagulation, so it lacks fibrinogen, coagulation factors and a lot of coagulation products. Fibrinogen can be converted into fibrin, which has the function of coagulation. Platelets secrete a large number of foreign bodies during coagulation. Some studies have found that nearly 50% of foreign bodies in serum come from extra secretion. Therefore, plasma is usually chosen in general experiments. In special cases, such as the study of platelet-related diseases, of course, serum is more suitable.
Limited by the sample, the blood sample is generally 2ml. What should I pay attention to when drawing blood? When studying blood samples from foreign bodies, a problem that is often overlooked is the size of the needle. In the process of blood drawing, when the blood flows through the needle, it will produce shear force. If the needle is too thin and the shear force is large, it will rupture cells or stimulate platelets to secrete exosomes, which will affect the follow-up experiments. It has been reported that 2 1 needle is more suitable for blood sample collection in exocrine research. Others, such as blood collection location, human physiological cycle, diet cycle and time cycle (24h), will affect the exosomes in the sample. So when sampling, try to be fixed and timed. Finally, it should be noted that the sample should be processed within 30 minutes after blood collection, because blood cells and platelets will also secrete exosomes when blood is collected in the collection tube.
4, runny nose
The study of nasal mucus has a special value, which can explore therapeutic foreign bodies. What is a therapeutic foreign body? In the treatment of brain diseases, drugs can be injected into the nasal cavity as exosomes to avoid the blood-brain barrier. The collection of nasal mucus is related to many environmental factors, such as age, season, sex, city, allergic history, occupational risk and so on. There is no uniform standard for collecting nasal mucus, as long as all samples are sampled in the same way.
Step 5 pee
In urine sampling, besides the influence of sex, age and diet, there is also a special point that the freshness of urine refers to the time when urine exists in the bladder.
6. Milk
Foreign bodies and antibodies in milk will affect the development of newborns and have great research value. The stage of lactation, parity, diet, milk yield, lactation, mother's health and mental state will all affect the secretion of exosomes. It should be noted that milk contains a lot of lipoprotein, which will affect the separation of exosomes in the later stage.
7.Cerebrospinal fluid
The difficulty of cerebrospinal fluid research mainly lies in the acquisition of samples, and the lack of exosomes in the samples makes it worse. Then how to solve this problem? Commercial normal samples can be purchased abroad, and other disease samples can also be used as controls.
Second, the preservation of foreign bodies.
For the preservation of exosomes, like other molecular samples, the extracted exosomes can be preserved at low temperature, which can be kept at 4 degrees for a short time (within one week) and at -20 or -80 degrees for a long time. Some people think that it is better to put it at 4 degrees, fearing that freezing and thawing at low temperature will affect the integrity of exosomes, but there is no conclusion yet, and most documents think that it has no effect. How many degrees should it be put? There is a trick, subcontracting -20 or -80, whether it is appropriate.