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Spinning the cells for 15 min at 350 x g has been proven and tested by PromoCell Research & Development. The QC department uses these settings during testing. Any lower centrifugation value (g-force and/or time) will lead to significant cell loss by means of non-sedimented; but intact; macrophages.
a) We usually perform macrophage differentiation in T75 flasks and 6-well plates. We haven't tested differentiation in smaller formats. But we assume it will be problematic to thoroughly wash the surface of the wells to remove non-adherent cells after the attachment phase. b) Detachment of the mature macrophages is possible but re-attachment can lead to significant cell loss (30-50%).
Please also keep in mind that working in 96/384 well-format has some inherent drawbacks (e.g.; evaporation of media; dry wells; etc.).
The Macrophage Detachment Solution (C-41330) directly affects the cell membrane. HSA in the Wash Buffer supports regeneration of the cell membrane and protects the cells during the critical phase directly after detachment from detrimental effects.
- FBS (or BSA) can be substituted for HSA; however we do not recommend this as the FBS will lead to unpreferable immunologic stimulation of human macrophages.
- The addition of EDTA to the PBS is to further augment the “anti-clumping” activity of the Ca2+/Mg2+ free PBS.
The source is porcine pancreas.
Most primary cells detach within 5-10 min at 37°C. Inactivation isn't required but we recommend to centrifuge the cell suspension to remove accutase and EDTA before replating the cells.
Please find attached a trouble shooting guide to identify possible reasons for poor detachment during subculture.
You can use our standard DetachKit (C-41200) to subculture the human nasal epithelial cells. Some customers still prefer to use our DetachKit-2 (C-41202) as it has a lower trypsin/EDTA concentration but tests in our cell culture lab haven't revealed any adverse effects when using C-41200.
Short protocol:
- Wash the cells with sterile PBS or HepesBSS
- Add undiluted accutase to the culture vessel (2 ml per 25 cm2)
- Incubate at room temperature for 5-15 min or at 37°C for faster detachment
- When the majority of the cells has detached; centrifuge the suspension and resuspend the pellet in fresh medium. In most cases no additional washes or neutralization steps are required.