Hi everyone,
I’m performing Annexin V-FITC/PI apoptosis assays using DU145 prostate cancer cells. However, I’ve been struggling with detaching the cells without affecting membrane integrity.
I use 0.25% trypsin-EDTA for 3 minutes, but DU145 cells often do not detach well. If I tap the plate or pipette repeatedly, I end up with lower viability and higher PI positivity, likely due to membrane damage.
I'd appreciate any advice on:
What detachment method works best for DU145 in apoptosis assay?
Have you successfully used low-trypsin?
How do you preserve cell membrane integrity during detachment?
Thanks in advance for your help!
Hello Anis Nadia
You should use accutase for detaching DU145 cells because accutase is known for its ability to maintain the integrity of cell surface antigens during detachment, which is crucial for accurate flow cytometry analysis. It also helps to minimize cell clumping, leading to a more homogenous cell populations and better flow cytometry data. Moreover, accutase often doesn't require a neutralizing solution after detachment, simplifying the workflow.
Accutase is typically used as a ready-to-use solution at its supplied concentration. Use enough accutase to cover the cells completely. Typically, 2.5 to 5 mL for a T-25cm^2 flask or 10 mL for a T-75cm^2 is recommended. Incubate for 5 to 10 minutes at room temperature. However, for faster detachment, incubate at 37°C. Check the flask frequently during incubation to observe cell detachment. Once cells have rounded up, tap the flask gently to dislodge any remaining attached cells. Please note that accutase need to be warmed up before use. You should rinse with PBS before adding accutase as it can be helpful. Neutralization is not required with accutase. Simply adding fresh media after detachment is sufficient to dilute the enzyme and stop its activity.
Best,
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