Isolation and Culture of Primary Neurons and Glia from a Rat Urinary Bladder
Transkript
Begin by placing rats on a sterilized surgical towel and exposing the abdomen. Use scissors and forceps to open the abdominal cavity and reveal the bladder. Use another set of scissors and forceps to gently lift the bladder and cut it from the bladder neck.
Then, quickly place the bladder in cold, oxygen-stable Krebs solution to improve cell survival. Add Krebs solution into three glass dishes and three glass beakers and place them in an ice bath for pre-cooling. Number the dishes and beakers 1, 2, and 3 to prevent confusion.
In glass dish 1, open the bladder with ophthalmic scissors and unfold it with forceps and a spoons nucleus divider. Rinse the bladder in glass beaker 1 and place it in glass dish 2. Eliminate adherent fat on the tissue surface using the forceps and ophthalmic scissors. Then, rinse the bladder in beaker 2 and place it in dish 3.
Gently scrape the bladder using the forceps and the spoons nucleus divider to remove exogenous attachments. Rinse the bladder in glass beaker 3 and transfer it to a 15-milliliter centrifuge tube with 14 milliliters of cold Krebs solution. Centrifuge the sample for 1 minute at 360 times g and 4 degrees Celsius.
Transfer the bladder from the centrifuge tube to a 2-milliliter vial containing 1 milliliter of digestion solution. Use ophthalmic scissors to cut the bladder into small pieces. Mix the bladder solution with 9 milliliters of digestion solution 1 in a sterile cell culture dish. Perform the first digestion in a shaking incubator for 1 hour at 37 degrees Celsius and 5% carbon dioxide at 200 RPM. After the first digestion, centrifuge the solution at 300 times g and 4 degrees Celsius for 8 minutes.
Meanwhile, place digestion solution 2 in a 37-degree Celsius water bath for preheating. After centrifugation, remove most of the supernatant that contains digestion solution 1, leaving some supernatant to avoid cell loss.
Resuspend the cells in warm digestion solution 2 and shake the mixture while digesting in a 37-degree Celsius water bath for 5 minutes. Once digestion is complete, immediately deactivate the trypsin by adding 10 milliliters of cold rinse media. Centrifuge the cells at 360 times g and 4 degrees Celsius for 8 minutes. Then, remove as much supernatant as possible to eliminate all trypsin.
Gently resuspend sediment with 3 milliliters of neuron media, making sure to not generate air bubbles in the solution. Filter the suspension through a 70-micrometer cell strainer into a 50-milliliter centrifuge tube. Collect cells by centrifugation at 360 times g at 4 degrees Celsius for 8 minutes. Then, gently resuspend the cell pellets in 1 milliliter of neuron media A and add 500 microliters of cell suspension to each well of a 48-well plate. Culture cells at 37 degrees Celsius and 5% carbon dioxide.
