I mechanically disrupted renal tumor tissue and then froze it at -70 for 1 month in DMSO 10% (in RPMI). Then samples were thawed at room temperature, centrifuged, resuspended in flow cytometry buffer FBS 1%, stained with antibodies and resuspended in flow cytometry buffer, fixed with PFA1%, and then analyzed by flow cytometry. Samples were analyzed in a Facscanto cytometer. I could only obtained dead cells.
Try infiltrating tumor with digestion buffer (collagenase/DNase in HBSS) using syringe and 30G needle, then cut into small pieces (~3 mm) with scalpel, digest at 37C for about 30 min. If you have access to Miltenyi GentleMACS tissue dissociator - try it, it really helps to have a consistent result (contact your local Miltenyi representative, they will help you to find the right dissociation protocol). After digestion/dissociation, pass cells through the 40 um nylon filter, lyse red blood cells (if tumor is vascular), and then cryopreserve cells (try fetal calf serum instead RPMI). Use isopropanol chamber of something similar for freezing cells. High number of dead cells could be result of incorrect sample dissociation, freezing or thawing.
Also, how did you do live/dead discrimination on the fixed cells? Did you do fixable live/dead stain first and then fixed your cells?
Hi, Evangelina
We have designed an antibody panel for diagnosis of pediatric solid tumors by flow cytometry, including the Wilms`s tumor.
You can find the complete non-enzymatic disaggregation methodology in the paper below.
Best regards,
Article Contribution of Multiparameter Flow Cytometry Immunophenotyp...
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