Hello all,

I recently started submitting neuroblastoma mouse tumor samples for single-cell RNA sequencing but am facing a huge problem with my tumor digestion being extremely gluey and slimy. The tumor digestion protocol I am using has been working well in the lab for the past few years, but right before I joined the lab, it stopped working.

I usually collect 800~900 mm^3 tumors, manually mince them with blades, and add the digestion buffer cocktail for enzymatic digestion. I have no problem processing the tumors until this step. After incubating the minced tumors for 30 minutes at room temperature on a rotator, I have extremely slimy and gluey tumor digestions where I cannot pass them through the 70 µm cell strainer. Even if I force them through, I don't get any pellet after centrifugation.

Here is my digestion cocktail recipe:

In 15mL polystyrene conical tube place 5 mL sterile HBSS on ice. Add the following (5mL total volume per tumor): 

75uL Collagenase IV (34mg/mL, final Conc.: 0.5mg/mL) 

50uL Hyaluronidase V (10mg/mL, final Conc.: 0.1mg/mL) 

150uL Dispase II (32mg/mL, final Conc.: 0.6U/mL) 

5uL DNAse I (Final conc: 0.005MU/mL) 

Has anyone had this problem of gluey and slimy tumor digestion?

Thank you for the advice and input in advance!