Hello everyone, I'd like to inquire whether frozen tissues are suitable for use as samples in flow cytometry experiments?
Your insights and experiences are highly appreciated.
Ideally no. Freezing alters surface protein conformations to a considerable extent. This may interfere with the binding of antibodies.
Hello Maryam Soltani Asl
I agree with Soumyadeep Mukherjee . You cannot freeze the tumor tissue for flow cytometry analysis. You may cut out many pieces of the tumor tissue and transfer them to a 50 ml tube containing MACS buffer (1X PBS, 0.5% FBS, 2 mM EDTA) for subsequent flow cytometry analysis. You may store the tube at 4°C overnight or use the tissue for flow cytometric staining immediately following digestion into a cell suspension.
There are several digestion methods available. Ensure that you choose the appropriate method (namely, that method which will preserve cell surface protein expression, viability, etc) for your downstream application. After having performed the right digestion step, filter the tumor cell suspension through a 70µm cell strainer. Centrifuge cell suspension at 250 x g, 4 °C, 5 min. Resuspend cell pellet in 1 ml FACS buffer (containing 1-5% FBS in PBS along with approximately 1-5mM EDTA). It is important that you work fast and under cold condition. Count cells using a hemocytometer. Then proceed with the flow steps.
Best.
Malcolm Nobre Hello Malcolm, thank you for your answer and sharing of the protocol.
- Badges
- Science topic
- Similar topics
- Philosophy
- Philosophy Of Science
- Reasoning