0, 0, 0 0. This procedure begins with isolating the hippo Campi from P zero newborn rats. Each brain has two of these structures, one in each medial temporal lobe.
Several hippo campi are collected in a 15 milliliter tube and associated with trypsin to a single cell suspension. Cell count and viability are determined using a hemo cytometer and the trian blue exclusion method. The primary hippocampal neurons are then plated on individual cover slips in a Petri dish.
Hi, my name is Joseph Nunez. I'm a faculty member of the neuroscience program at Michigan State University. Today I'll be demonstrating a typical hippocampal culturing protocol.
Before we begin the hippocampal isolation, we need to make sure that all the tools are sterile. Spray down the culture hood with 70%ethanol and place the tools inside the hood. You'll need six and 10 centimeter Petri dishes.
Sterile poly, L coated glass cover slips, pipetters, and tips, disposable pipettes, and an electric pipetter. From this point on, remember to use correct sterile technique, turn on the water bath and make sure that it is heated to 37 degrees Celsius. We will also need the following solutions that are stored at four degrees Celsius, modified eagles medium, or MEM neuro basal hank's buffered saline solution, bore buffer solution, sodium pyruvate solution and sterile filtered 20%glucose solution in I distilled water.
Take these frozen solutions outta the freezer and place them in the water bath. A five milli aliquot of horse serum. A one milliliter aliquot of B 27 supplements a one milliliter aliquot of a hundred times antibiotic, which includes penicillin plus streptomycin, and a 0.5 milliliter aliquot of L-glutamine.
Place all of these solutions in the heated water bath. Take a 15 milliliter conical tube, fill it with HBSS and label it with the treatment group. In sex, it is here that the isolated hippo Campi will be collected in the next step.
To begin the hippocampal isolation, make sure postnatal day zero newborn rat pups are clean and have had their milk bands, placenta and umbilical cords removed by their mothers. Remove the head from the body with scissors using the same scissors cut through the skin and the skull. Using a pair of fine tweezers, peel the skull away from the brain and place the brain into a small Petri dish that contains a small amount of sterile HBSS.
Peel back the cerebral hemispheres. The hippocampus is a small seahorse shaped structure in the medial temporal lobe. Remove the hippocampus and place into three milliliters of HBSS in a 15 milliliter tube.
Repeat these steps with each pup and place each isolated hippocampus into the 15 milliliter tube. Since today, we'll be plating cultures on six cover slips. We have collected 12 hippo Campi from six rats in this tube.
Now we are ready to dissociate the tissue to single cells. After all, hippo Campi have been isolated, fill the 15 milliliter tube to 4.5 milliliters with HBSS. Add 0.5 milliliters of trips into the tube and incubate for 15 minutes at 37 degrees Celsius.
Remove the HBSS solution from the tube with the sterile pipette. Being careful not to disturb the hippocampal that have settled to the bottom of the tube. Add five milliliters of HBSS to the tube and swirl gently incubate at 37 degrees Celsius for five minutes.
Repeat this step twice. Removing the old HBSS and replacing with fresh solution as 0.5 milliliters of D Ns to the Hippo Camp Eye in 4.5 milliliters of HBSS, the DNAs one is added to promote enzyme inactivation. Iterate the solution or pipette up and down vigorously until it is homogeneous.
Be careful not to introduce bubbles into the homogenate. Next, determine cell viability with the Trian blue exclusion method. Take a 10 centimeter Petri dish containing six eight cover slips and add 10 milliliters of plating.Medium.
Pipette the desired number of cells to the dish containing the cover. Slips allow the cells to attach for two to four hours in a humidified 37 degree incubator with 5%CO2. After confirming that the cells are viable and have attached transfer the covers lips to individual dishes containing neuro basal medium, and place these dishes into the incubator.
Once a week, we replace one third of the medium with fresh neuro basal, medium, and and experimental groups as needed. Now we're ready to study the functional properties of these cells in culture. After cultured, hippocampal neurons have been in vitro for 48 hours, but no earlier.
We are ready to begin calcium imaging experiments. At this point, these neurons should have begun to extend processes. The optimal age range for calcium imaging is from day in vitro.
Three to seven cultures are loaded with URA 2:00 AM for 30 minutes at 37 degrees centigrade, at which time they can be imaged under a 20 times objective. Using using a xenon arc to excite the calcium indicator dye and a five 12 bit CCD camera, which samples every 150 milliseconds in this time. Last movie of calcium flux, our hippocampal neurons have shown musca mol and glutamate induced calcium responses.
Today we'll demonstrate how to perform hippocampal cell cultures and just make sure to use proper sterile technique and you'll be able to culture in up to 20 minutes for a litter of six to eight animals. Well, that's it. Good luck with the experiments.
