Hello Feiyi Chen

The delicate nature of neurons and the tough connective tissue of the DRG present significant challenges, so careful and gentle handling is crucial to maximize the number of viable, single cells.

I would suggest you do the following.

1. If you use young animals, it will help. The DRG from younger mice (for instance, 5-8 weeks old) contain less connective tissue and are much easier to dissociate, resulting in a significantly higher yield of viable cells. Older animals have toughened connective tissue that requires more aggressive enzymatic and mechanical dissociation, which can harm cell viability.

2. You should combine mechanical dissociation and enzymatic digestion of the tissue.

3. Use an enzyme cocktail, typically containing a mix of collagenase and papain or dispase. Use fresh enzyme solutions to ensure maximum activity. Incubate at the recommended temperature (namely, 37°C) but avoid over-incubation as it can decrease cell viability.

4. Adding DNase I to the digestion mixture will be helpful. This is because when cells die, they release DNA, which makes the suspension clumpy. DNase I degrades this free-floating DNA and will prevent cell aggregation.

5. For mechanical dissociation, use fire-polished glass Pasteur pipettes with progressively smaller diameters to gently triturate the tissue. This will minimize damage from excessively aggressive pipetting. During this procedure avoid introducing air bubbles, which can harm the cells. At any time during the process avoid vortexing the cell suspension. This is aggressive and will kill many of the fragile DRG neurons.

6. For filtration of the dissociated cell suspension through a cell strainer, use 70µm, which is a good choice for obtaining single-cell suspension. A 140µm strainer is typically too large for filtering cells and is more suited for removing large tissue debris, not for routine cell dissociation.

7. Introduce an extra step namely, use a density gradient medium (such as Percoll) to separate viable cells from debris, myelin, and smaller non-neuronal cells. This technique will significantly improve the purity of the neuronal population. Neurons typically form a layer at the interface of the density gradient, while debris and non-neuronal cells remain in the upper layer.

8. After enzymatic digestion, you should immediately add a serum-containing medium to inactivate the enzymes and protect the cells.

9. Finally, if you must increase the number of cells, try to minimize cell loss. For this purpose, utilize low-protein binding tubes and plates to prevent cells from sticking to surfaces and being lost. Store dissection media and tissue on ice whenever possible before enzymatic digestion to reduce metabolic stress on the cells.

Best,