The overall goal of this procedure is to produce in vivo like astrocyte monocultures using a simple, fast, and economical method. This method can help answer key questions in the astroglibiology field, such as what are the economics of astrocyte physical release or uptake. The main advantage of this technique is the resulting cells closely resemble astrocytes in vivo by morphology, mRNA and protein expression, and intercellular calcium fluctuations.
To begin this procedure, prepare the dissection area by placing two 10 cm petri dishes filled with dissection medium on ice in a laminar flow dissection hood. For each brain area to be isolated, prepare a 15 mL tube filled with 14 mL of dissection medium and keep on ice. Next, spray the dissection tools with 70%ethanol and place them next to the dissection microscope in the hood.
If working with embryonic tissue, first remove the embryos from the uterus and open the embryonic sacs. Immediately, cut off the heads with scissors. When all heads are cut off, transfer them into pre-cooled dissection medium.
Once the heads are submerged in the medium, open the skin and skull with forceps and pinch off the cerebellum. Then carefully lever the rest of the brain up and out of the skull. After that, separate the cortex from the underlying mid-brain structure.
Place the forceps within the longitudinal fissure between the hemispheres and move them in between the cortex and mid-brain structures of one hemisphere to pinch each hemisphere free. Subsequently, remove all meninges from each hemisphere and separate the hippocampi. If the hippocampal astrocytes are required, transfer each hippocampus into a 15 mL tube filled with 14 mL of dissection medium on ice.
After that, cut any cortical tissue to be used for generating astrocytes into pieces no larger than one cubic milliliter. Then collect all pieces of cortical tissue in a separate 15 mL tube filled with dissection medium on ice. Once all tissue pieces are collected, wait for them to settle to the bottom of the tube.
Then aspirate as much medium as possible. Following that, add 2-3 mL of 0.05%tripsen EDTA and incubate the samples in a 37 degree Celsius water bath for twenty minutes. Afterward, carefully aspirate the tripsen EDTA leaving tissue pieces in a minimum amount of liquid at the bottom of the tube.
Next, add 5 mL of pre-cooled dissection medium to wash off the remaining tripsen EDTA and pipette to the side of the upper tube to achieve mixing of the tissue pieces. Then, aspirate the dissection medium and leave the tissue pieces in as little liquid as possible. Repeat this washing step with pre-cooled dissection medium twice.
After the final washing step, add 1 mL of room temperature D-Mem Plus, and triturate the tissue by pipetting up and down without introducing bubbles. Astrocytes are quite sensitive to pH, so it's important to avoid producing bubbles as this may change the pH of the solution. Next, place a 100 micron cell strainer in the opening of a 50 mL tube and pre-wet the filter with 4.5 mL with pre-cooled D-Mem Plus.
Pipette 1 mL of the dissociated tissue suspension onto the cell strainer and add another 4.5 mL of pre-cooled D-Mem Plus to wash the cells through it. On day in vitro seven after the dissection, place the 10 cm dishes with cells in D-Mem Plus on a shaker in the incubator and shake the dishes at 110 RPM for 6 hours. 20 to 30 minutes before the end of the six hours shaking, pre-warm 1 XPBS, D-Mem Plus, NB+H, and 0.25%tripsen EDTA to 37 degrees in the water bath.
After 6 hours of shaking, take the culture dishes off the shaker and immediately replace the medium with 10 mL of pre-warmed 1 XPBS per dish. Subsequently, removed PBS and add 3 mL of pre-warmed tripsen EDTA per dish. Then incubate the samples for four minutes at 37 degrees Celsius.
If preparing samples for Western Blot or RNA sequencing, do not add Tripsen EDTA. Instead, replace the PBS with 12 mL of pre-warmed NB+H, after which cultures can be placed back in the incubator. Next, add 5 mL of pre-warmed D-Mem Plus to 3 mL of tripsen EDTA in each dish.
Pipette the cells off the dish and collect the cell suspension in a 50 mL tube. After that, centrifuge the cell suspension at 3220 times G at 20 degrees Celsius for 4 minutes. Then, remove the supernatant and re-suspend the cell palate in 1 mL of pre-warmed NB+H.
These images show that G-camp transduced ausam astrocytes exhibit spontaneous calcium ion events throughout the astrocyte networks including in the thin processes. Here, a calcium ion wave travels through several astrocytes from left to right in the images of distinct time points, where light color depicts areas of high-calcium ion concentrations and black areas represent extra cellular regions. In this graph, calcium ion concentration changes in the color-coded regions over time, which are shown as G-camp fluorescent intensity traces, illustrating how a calcium ion signaling wave spreads across several astrocytes.
Once mastered, this technique can be done in one hour and fifteen minutes if it is performed properly. Following this procedure, other methods like life cell imaging in combination with transfection or viral transduction experiments can be performed to answer additional questions like which events occur in astrocyte membranes. After watching this video, you should have a good understanding of how to prepare in vivo like astrocyte motor cultures in a simple, fast, and economical way.
