Using 1x trypsin for 5 minutes did not work. Then we incubated for 20 minutes but still few cells detached.
Hi,
I hope the reference (Article Impact of Detachment Methods on M2 Macrophage Phenotype and Function
) is useful for your reference. Please note that "while enzymatic methods provide efficient macrophage detachment, they alter the level and function of specific cell surface molecules. While treatment with EDTA or PBS on ice required longer incubation times and partially reduced cell viability, within the constraints of the markers evaluated, they did not alter cell phenotype and function. As such, in vitro analysis of M2 associated phenotype and function, which requires detachment, is best studied using EDTA on ice. In contrast, accutase and trypsin based detachment solutions could be utilized when studying the FcγRs and antigen presenting markers along with their associated functions."Therefore, depending on the type of experiments you are conducting, you can choose a suitable method...
Good luck!
Hi Javiera Cárdenas
0.25% Trypsin for 30 minutes at 37°C is a common approach used in many protocols. To refer to additional methods and approaches please find the attached link.
Article Impact of Detachment Methods on M2 Macrophage Phenotype and Function
Thanks,
The use of trypsin for the culture of human macrophages can vary depending on the specific protocol, cell line, or experimental requirements.
1. Trypsin Concentration: Low concentrations ranging from 0.05% to 0.25% are typically used, though higher concentrations may be necessary for strongly adherent cells.
2. Incubation Time: Varies from a few minutes to 10-15 minutes depending on cell adherence and experimental conditions. Macrophages, being adherent, may require longer incubation.
3. Temperature: Trypsinization is conducted at 37°C, the physiological temperature for mammalian cells, though deviations may occur based on experimental needs.
4. Buffer: Trypsin is often used with buffers like PBS or HBSS to maintain osmolarity and pH during detachment.
5. Neutralization: Neutralization of trypsin activity post-treatment is crucial to prevent cell damage, typically achieved by adding a trypsin inhibitor or serum-containing medium.
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