Hello everyone, I've been trying to find an article of a standarized protocol for subculturing THP-1 macrophages, however I haven't found anything about that, does anyone know what is best for detachment? I'm using 5 ng/ml of PMA to differentiate THP-1 cells and giving them 72hrs to rest, I want those cells for a co-culture with A549 cells.
Hello Daniel Ortiz Ramírez
Macrophages are difficult to detach as they get very sticky. If you use aggressive methods of detachment, there is a risk of damaging the cells. One possible way would be to cultivate macrophages on non-cell culture treated substratum like using bacterial plastic, or you could refer to the research article attached below for the detachment methods. Please refer to the Materials and Methods section (2.2) of the attached paper.
Article Impact of Detachment Methods on M2 Macrophage Phenotype and Function
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2.2. Detachment Methods
All reagents were purchased as ready-to-use solutions, and minimal volumes sufficient for coverage of growth surfaces were employed. For enzymatic methods, cells were incubated at 37 °C with either 0.25% trypsin containing 0.9 mM EDTA (Invitrogen, CA, USA) or Accutase (Biolegend, CA, USA) for 5, 10, 20, 40, 60 minutes, gently pipetting until cells detached. Non-enzymatic methods included incubation of cells with phosphate-buffered saline (PBS) or 5 mM EDTA (Quality Biological, MD, USA) on ice for 20, 40, 60 minutes with more vigorous pipetting. At the time points indicated, trypsin, Accutase, PBS, and EDTA activity were neutralized with RPMI-1640 complete medium. Macrophages were also detached by gentle scraping with cell scrapers (Sarstedt, NC, USA). After detachment, cells were washed 3 times with PBS supplemented with 2% FBS and centrifuged for 5 min at 900xg.
Best.
Dear @Danial, Depending upon your downstream purpost you must choose methods of detachment. If you want to isolate RNA then directly incubate cells with trizole and scrape it. But if you want to analyze cells then you must choose Accutase treatment at different time points (need to optimize in your laboratory), with intermittent tapping of flask, still you see cells adhere to surface, you may use cell scrapper gently or vigorous pipetting to dislodge the macroophages.
Remember that detachment of macrophages is much difficult task then making MDMs.
Good luck.