In my study, I intend to infect PBMCs with SARS-CoV-2. After that, I will analyze NK cells by flow cytometry to see if their phenotype changes or if they show degranulation.
After the infection, I need to perform washes. I will aspirate the entire supernatant from the plate and transfer it to a 1.5 mL tube, then centrifuge it. After that, I will discard the supernatant and resuspend the cell pellet before placing it back into the plate. 24 hours after infection, I will collect the total supernatant and analyze the NK cells by flow cytometry.
Since I have never worked with PBMCs before, I have some questions:
PBMCs are a mixture of immune cells, including lymphocytes (T cells, B cells, and NK cells) and monocytes, which can be susceptible to viral infections. The specific virus that infects PBMCs depends on various factors, such as:
Virus tropism (the ability of a virus to infect specific cell types)
Host immune status
Exposure to the virus
Hello Felipe Costa
The answers are as follows:
1. When aspirating the supernatant from the plate and transferring it to a tube, and then centrifuging for 10 minutes, the cells adhered to the plate will be left without culture medium. Could this kill the adhered cells?
Yes. You should add fresh culture media just enough to immerse the cells. As far as possible avoid exposure of cells to air. This could have a damaging effect on the cells.
2. What would be the ideal speed, time, and temperature for centrifugation?
Ideal speed: 1200 – 1500 rpm
Ideal time: 10mins
Ideal temperature: 25 ± 2°C
3. For washing the cells, what is the ideal temperature for PBS?
For washing the cells, the ideal temperature for PBS is room temperature (i.e., 25 ± 2°C).
4. What is the recommended maximum time to extract PBMCs after blood collection in an EDTA tube?
Isolate PBMCs within 4-6 hours after blood collection to maintain viability.
5. After isolating PBMCs, I will place them in plates with RPMI medium. I will only perform the virus infection the next day. Is there any problem with this?
No problem! Before using the cells in any cell-based assays, it is recommended that you rest the cells overnight (8-16 hours) in culture media at 37°C in 5% CO2 incubator.
Good Luck!
Considering that you don't seem to have enough experience and maybe you don't use the right facilities (e.g., BSL3 lab), please stop your activity on this mutating virus which has caused the death of millions of people.
Alireza Naderi Sohi
My dear, I perform all my procedures with viruses in BSL3. Rest assured. I have never worked specifically with PBMCs, but I have worked with other types of cells. Various types of cells.
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