HI,
I'm currently working on adherent human epithelial cells.
In ordrer to evaluate the apoptosis effect of a drug, the cells need to be cultured in a FCS-free medium before the addition of the drug in this same medium.
After cell detachment from their support (plastic) by trypsin, they are washed with specific annexine V binding buffer (BD Bioscience Kit) and incubted with the apropriate amount of annexin V-FITC and IP before flow cytometry according to manufacturer's instructions.
In all experiments, the level of annexine V/IP is high (double labelling) and not modified by cell treatment.
I have no explanation about this fact.
can you help me?
Many thanks
Hi Olivier,
Have you checked the viability of cells before flow cytometry, with neubauer cell counting with trypan blue?
Your cells may be susceptible to your detachment method or high mechanical stress. Sometimes, washing the cell layer with PBS (w/o Mg/Ca) twice before the addition of trypsin already helps improve the efficiency. Furthermore, you could try to use lower concentrations of trypsin and/or lower incubation times. Additionally, milder alternatives should be tried out, e.g. TrypLE or accutase. Also try to avoid very high mechanical stress, e.g. use 1000 µl tips instead of 100 µl tips for resuspension.
Another reason for high levels of Annexin V/PI-positve fractions under any conditions could be an inappropriate compensation or too high voltages for your detection channels. Define the voltage range with (1) unstained, (2) untreated + (single) stained and (3) treated + stained cells (positive control, e.g. treatment with doxorubicin). Furthermore, make sure to use appropriate gates that exclude debris and cell aggregates from the detection.
After checking all above-mentioned notes, you should be able to discriminate between vital, apoptotic and late-apoptotic/necrotic cells.
Hi Dr Olivier,
I agree with Dr Lange. I also had the same problem before. The way that I solved this problem is to add serum-containing medium immediately to each well after trypsin incubation (try the shortest time in trypsinization because trypsin is also toxic to cells) and then gently collect the cells in each well.
Hope it is useful
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