Hello dear scientists.
I have a question regarding the EOC-20 microglia cell line. I will perform cell culture with this cell line and then ICC for the first time. I do not have a proper protocol, and I've searched and read a lot but can't find a detailed protocol in that regard. for example, I need to have a culture schedule that explains when I have to split cells and so on...
I appreciate any help in this regard.
Thank you a lot in advance.
According to ATCC, you should culture these cells in a conditioned DMEM which contains colony stimulating factor 1 (CSF-1). They cite using LADMAC cells to produce the conditioned media supplement. They subcultivate 1:3 every 2-3 days.
Zahra Farhadi, You need to culture those cells in the complete DMEM media supplemented with10% FBS. Then You have to incubate them in standard sterile culture conditions. After 3-4 days, you will check your cultured cells for confluency through microscopy. If up to 80% of cells will be confluent, you will subculture them either by trypsinization or gentle scraping in the laminar hood. Generally, the passaging ratio is 1:3, which means 1 part of your confluent cultured cells and three parts of fresh complete culture media. Then you will seed them in three T25 flasks. The initial volume should not be more than 5 ml in each T25 flask.
For Immunocytochemistry, You have to grow cells in gelatin-coated sterile coverslips in 6/24-well plates. As the cells achieve the desired density, you will replace culture media by washing with 1xDPBS, followed by fixation in 2-3% paraformaldehyde for 15 minutes at RT. Then you will wash these wells twice with DPBS or wash buffer. In this way, you can use these fixed cells for your desired immunofluorescence staining experiments.
I hope this will help you.
@Vinit Sharma Thanks a lot for your help :). Yes it is indeed very helpful.
So, I first culture my cells in flasks then for ICC I will transfer the desire volume to 6/24 well plates? Is that correct?
Zahra Farhadi, Yes, you have to do it in a similar way.
For more details on immunocytochemistry, you can check the below-mentioned Link and standardize your experiment accordingly.
https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-icc-staining-cells-coverslips
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