This protocol provides a reliable reference for preparing a high-quality single cell suspension from the Nile tilapia intestine to facilitate single-cell level studies in aquaculture fish species. The technology is highly efficient and can be implemented in most laboratory conditions. It reduces the need for additional trials to prepare single-cell suspensions for aquaculture fish species.
Begin by rinsing six month old Nile tilapia fish in well aerated sterile water for 15 minutes to wash off loosely bound bacteria from the surface. For preparing the enzymatic cell dissociation reagent, dilute the collagenase dispase in PBS to a final concentration of one milligram per milliliter. Mix 95%volume of the prepared enzymatic solution and 5%volume of FBS.
Then pre-cooled the centrifuge to four degree Celsius before use. For tissue dissection, place the euthanized fish on ice and collect three to four centimeters of the intestines midsection. Excise the fat and mesentery using forceps.
Remove the fat attached to the intestinal surface and the elastic and malleable clear mucosa. Gently rinse the intestine fragment with sterile ice cold PBS using a syringe to wash off the intestinal contents and mucus. After several washes, dissect the fragment into small pieces and transfer them to a 1.5 milliliter tube containing one milliliter ice cold PBS.
Centrifuge the mixture at 300 x g in four degrees Celsius for five minutes. And replace the supernatant with one milliliter of ice cold 0.08%BSA-DPBS. Using a glass pipette gently aspirate to resuspend the tissue fragments.
After washing the pieces one more time, remove the supernatant and add one milliliter of the prepared enzymatic dissociation reagent. Also mix five microliters of RNAs inhibitor with the dissociation reagent for single-cell RNA sequencing. Place the tube at 37 degrees Celsius tempered water bath and manually invert the tube every five minutes during the 30 to 60 minutes incubation time.
Once the tubes are spinned, remove the enzymatic cell dissociation reagent using wide-bore tips. Add one milliliter of ice cold 0.08%BSA-DPBS and gently pipette the cells up and down to prevent cell disruption. After centrifusion and resuspending the cells one more time, place the 40 micrometer cell strainer pre-wetted with 0.08%BSA-DPBS onto a 50 milliliter conical tube.
Pass the cell suspension through the strainer. Tap the strainer, then rinse it with 200 microliters DPBS and collect the suspension at the bottom of the tube. After transferring the suspension into a 1.5 milliliter tube, add five microliters of DNAs and incubate the suspension for 15 minutes at 15 to 20 degrees Celsius.
Once centrifuged, remove the supernatant and resuspend the cell pellet in 400 microliters of ice cold DPBS without calcium and magnesium ions. Then gently pipette up and down with wide-bore tips to mix the cells and repeat this process one more time. For staining, mix the cell suspension in 0.4%trypan blue solution in an equal ratio and incubate for three minutes at room temperature.
Then add 10 microliters of the mixture on the glass slide. Count the viable and dead cells under the microscope in three fields and calculate the cell viability percentage. The dissociation efficiencies of several commonly used enzymes were compared.
The collagenase dispase mix was verified to have a better dissociation effect than the collagenase dispase or trypsin alone and several other enzyme mixtures. The microscopic examination showed that the intestinal cells had high viability and dispersed as single cells. Most cells were viable, whereas a few were stained dead because of the permeablized cell membrane.
Pay attention to the type of PBS used. Enzymatic digestion solution cannot be used with calcium/magnesium free PBS as the enzyme requires calcium/magnesium ions to function effectively. The procedure facilitates single-cell level studies in aquaculture fish species providing a valuable reference for developing cell dissociation protocols of aquaculture fish species.
