This article details a protocol for isolating hippocampal cells from adult mouse tissue using enzymatic dissociation and density gradient centrifugation. The method aims to obtain viable neurons and glial cells for further analysis.
Take perfused adult mouse hippocampi fragments in a dissociation tube containing papain and DNase.
Place the tube on a mechanical dissociator.
The dissociator mechanically disintegrates the tissue, loosening cells, including neurons and glial cells.
Papain breaks down the tissue's extracellular matrix, releasing cells. DNase degrades contaminating DNA.
Add buffer containing BSA, inactivating the enzymes.
Pass the suspension through a strainer to remove large debris.
Centrifuge the filtrate. Discard the enzyme-rich supernatant.
Resuspend the cells in buffer, and transfer them to a fresh tube.
Add density gradient media, mix, and overlay the mixture with buffer.
During centrifugation, cells and debris form separate layers based on their densities.
Remove the topmost buffer layer and the middle debris layer.
Add buffer to the bottom layer containing the cells and mix. Centrifuge and discard the supernatant containing the density gradient media.
Resuspend the hippocampal cells in buffer for further analysis.
Using forceps, transfer the hippocampi tissue pieces to a C-tube, and add 30 microliters of enzyme mix 2 into the tube. After twisting the cap and tightening until it clicks, place the tube in a dissociator, and run the appropriate program. While the program is running, pre-wet a 70-micron cell strainer placed on a 50-milliliter conical tube with 2 milliliters of BSA buffer.
After the dissociation program is complete, add 4 milliliters of BSA buffer to the dissociated tissue, and filter the mixture through the cell strainer on the 50-milliliter conical tube. Next, add 10 milliliters of DPBS to the C-tube. Then close the tube, swirl the solution gently, and filter it through the cell strainer on the 50-milliliter conical tube. Centrifuge the filtered cell suspension. Discard the supernatant without disturbing the pellet, and store the pellet.
For debris removal, resuspend the pellet with 1,550 microliters of cold DPBS and transfer the suspension to a labeled 15-milliliter conical tube. Then, add 450 microliters of cold debris removal solution, and pipette up and down. Gently overlay the cell suspension with 1 milliliter of cold DPBS, keeping the tip against the wall of the conical tube. Repeat the procedure until the total overlay is 2 milliliters.
Next, centrifuge the suspension at 3,000 times g for 10 minutes with full acceleration and full brake. Aspirate the topmost layer. Then, sweep the pipette tip back and forth to aspirate the white middle layer. Remove as much of the middle layer as possible without disturbing the bottom-most layer. Next, add 2 milliliters of cold DPBS, and pipette up and down to mix. Then, centrifuge the suspension at 1,000 times g for 10 minutes with full acceleration and full brake. After centrifugation, discard the supernatant, and resuspend the pellet in 1 milliliter BSA buffer.
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