In the first part of this video, we will demonstrate how to prepare the astrocyte neuron co-culture. To do this, several hippo Campi from P one rats are collected into a tube digested with propane, dissociated by ation, and then plated onto cover slips. The second section will show how to measure intracellular calcium dynamics in astrocytes, which involves loading of flu oh four calcium indicator dye into astrocytes and measuring global calcium dynamics by epi microscopy and measuring near membrane calcium dynamics by turf microscopy.
By changing the illumination automatically and quickly, both global calcium and near membrane calcium can be observed almost simultaneously. Hi, I'm Aji omi, a postdoctoral researcher from Budget COS lab in the Department of Physiology at the University of California Los Angeles. Today we'll show you a procedure for calcium imaging of asto sites.
We use this procedure in our laboratory study near membrane and global calcium dynamics in as osteocyte. By changing the elimination automatically and quickly, we can resolve global calcium and near membrane calcium. Almost simultaneously, I will show how we apply a TP to cause calcium elevation in astrocyte.
I will then finish by summarizing and discussing the barrier with the method for studying astrocyte. So let's get studied. To begin the astrocyte culture, remove the skin and skull from the head of a decapitated rat pup.
Place the brain in a Petri dish filled with cold dissection media, dissect both hemispheres and remove the meninges. Next, dissect the hippo campi. Chop the hippo campi into approximately one by one millimeter pieces.
Digest in Pappa solution at 37 degrees Celsius for 11 to 13 minutes. Remove the dissected tissue from the incubator. Then once the pieces of tissue have settled, remove them from the propane digestion carefully and add five milliliters of hippocampal medium to wash the chopped pieces of hippo Campi.
Repeat this step and then finally, resus suspend in hippocampal medium. Iterate the cells about five times with three flame polished progressively smaller pipettes. Once ated passed the cells through a cell strainer with a whole size of 70 micrometers and add four milliliters of medium to the strainer.
Count the cells in 10 microliters of suspension. Adjust the volume of hippocampal medium in order to have 400, 000 cells per milliliter. Now prepare cover slips for seeding the cell suspension by aspirating laminate from the cover slips, which have been exposed to a laminate solution overnight.
Before the covers can dry plate 200 microliters of the cell suspension per cover slip. Leave them to attach for 60 minutes in the incubator. After incubation, add two milliliters of hippocampal medium per well.
The next day aspirate the old hippocampal medium to remove dead cells and debris and add two milliliters of pre-warned fresh hippocampal. Medium maintenance of astrocyte neuron Co cultures should begin four days after plating and adding one milliliter of fresh neuro basal medium twice a week. Be sure to pre incubate the media about 30 minutes in the incubator in a ventilated flask to equilibriate the temperature and carbon dioxide.
Before studying the astrocytes, it is useful to look at nano fluorescent beads to optimize laser angle for turf. To do this, put 200 microliters of water on the Petri dish and add one microliter of 100 nanometer fluorescent beads and mix look at the beads by epi fluorescence microscopy. Next, look at the beads by turf microscopy.
For calcium imaging, place the culture in a well on a six well plate filled with two milliliters hippocampal recording buffer containing 2.5 micromolar flu oh 4:00 AM and 0.05%onic F1 27, 20%solution in DMSO and incubate at room temperature for 10 to 30 minutes, remember to cover the plate to avoid bleaching the fluoro. Four, remove the flu oh four solution and wash the cover Slips three times with hippocampal recording buffer. Incubate the washed cover slips at room temperature for 30 minutes.
Place the cover slip in the chamber, clean the other side of the cover slip with lens cleaning liquid and add 200 microliters of hippocampal buffer buffer. Put one small drop of immersion oil on the objective and put the chamber on the stage of the microscope. Look at the cells using transmission light to focus and see how the cells look.
Next, illuminate the cells with 488 nanometers from a monochrome and determine if the fluorescent signal of flu oh four is uniform and detectable. In astrocytes, use epi and turf illumination to measure calcium in the astrocytes. Here, fluorescent beads are observed by epi illumination.
In addition to producing background fluorescence, they also display significant brownian motion as the angle of illumination. With the laser approaches the critical angle, the laser beam is totally reflected from the glass water interface. This total internal reflection generates an electromagnetic field called the evanescent field.
This has a depth into the cell of lower refractive index of about 100 nanometers. You can excite floral fours in this thin layer on the specimen surface. Here are fluorescent beads observed by turf illumination when compared with epi.
Illumination signal to noise ratio significantly increases when you decrease the angle. The beam of the laser does not reflect at the surface and passes through the cover slip to hit the fluorescent beads directly. You can see a lot of background fluorescence.
Now we increase the angle. Again, background noise significantly decreases and you can see fluorescent beads near the surface of the cover slip. These images show flu oh four loaded astrocytes and neurons.
Astrocytes are flat cells with fine processes, whereas neurons have round cell bodies and thick and long dendrites. Both astrocytes and neurons look healthy and loaded with flu. Oh four properly.
Looking at astrocytes by epi or confocal microscopy is useful to know if astrocytes are healthy and if calcium indicators are loaded into astrocyte properly. In the regions of the astrocyte that are closest to the cover glass regions called the footprint, a TP is observed to cause a calcium increase near the plasma membrane. Hippocampal astrocytes are known to express pure anergic receptors and a TP when applied to the bath can cause a global calcium release.
We've just shown you how to prepare hippocampal osteocyte culture and how to measure the calcium level of as osteocyte both globally and near plasma membrane. When doing this procedure, it's important to remember to optimize laser angle for the best way to ensure this on the microscope is to observe the behavior of 100 millimeter flow speeds. The simultaneous recording of global calcium and near membrane calcium level will provide us not only detailed description of calcium signal, but new insight of the mechanism of calcium regulation in astrocyte.
It also occurred to us that the approach described here is useful for measuring near membrane and global intracellular calcium level in other excitable unknown excitable field. So that's it. Thank you for watching and good luck with your experiment.
