The overall goal of this procedure is to prepare primary cell culture of xenopus retinal cells. This is accomplished by first removing the surrounding tissues to expose the retina. The second step is to excise retinal tissue.
Next, the retinal tissue is dissociated into single cells. The final step is to plate retinal cells on an UnCloned dish and add flu oh 4:00 AM for calcium imaging. Ultimately, confocal microscopy is used to detect calcium activity in retinal cell culture.
This method can help to answer key questions in the fields of developmental biology, neurobiology, and cell biology, such as the role of calcium and electrical activity in retinal development, as well as gene expression on a single cell level. Generally, individuals new to this procedure will struggle because fine motor movements are necessary for timely and accurate dissections. Visual demonstration of this method is critical as the dissection steps are difficult because intricate hand-eye movements are essential.
To begin this procedure inside the hood, prepare and label the following, one 60 millimeter plastic Petri dish containing 10 milliliters. Cell culture medium to be used as a dissection plate. Optionally, 10 milligrams collagenase B can be added to the dissection plate for a final concentration of 1.0 milligrams per milliliter to aid in tissue layer separation for dissection of embryos at stage 25 or younger.
Also, prepare 1 35 millimeter lon surface dish containing two milliliters. Cell culture medium to be used as a culture plate. One empty 1.5 milliliter micro centrifuge tube to be used as an explan dissociation tube and one 15 milliliter conical falcon tube containing 15 milliliters.
Cell culture medium. Then prepare 2 35 millimeter plastic Petri dishes containing two milliliters, CMF for dissection, one to be used as an explan dissociation plate, the other as an X explan rinse plate outside the hood. Prepare two 60 millimeter plastic Petri dishes containing 10 milliliters.
0.1 XMMR, one to be used as a holding plate, the other as a sibling plate, and one more 60 millimeter plastic Petri dish containing 10 milliliters, 0.1 XMMR plus 0.5 milligrams per milliliter. MS 2 22. After that, add 40 microliters, 5%tripsin in CMF to the explan dissociation plate.
Swirl to mix, then set aside. Next, proceed with dissection using a dissecting scope, using two pairs of fine forceps. Make a mid sagittal cut on the ventral side of the embryo beginning from the posterior end, and continuing through the cement gland in the anterior portion of the embryo.
Carefully open the embryo by spreading both sides of the cut. This results in the dorsal side of the embryo facing the dissection plate. Then open the ventral ectoderm to reveal the endoderm and mesoderm.
Next, remove the endoderm and mesoderm the first and largest layer of this tissue. The endoderm is very fluffy in appearance with large cells and little obvious organization. After removing this layer, the somites and Noor will become visible.
For better contrast, transfer the embryo to a separate 60 millimeter plastic Petri dish containing 10 milliliters, cell culture medium, and 100 microliters of 1%solution of Nile blue sulfate and incubate for two to three minutes. Afterward, transfer it back to the dissection plate. This will stain the ectoderm, somites, and Noor for easier identification and orientation.
Focusing on the anterior portion of the embryo, carefully remove the Noor and any remaining mesoderm to expose the brain and optic vesicles. Once the brain and optic vesicles are completely exposed, use the forceps to sever the neural tube and the underlying ectoderm just posterior to the brain. Turn this portion over so that the ectoderm is on top.
Use caution to avoid damaging the underlying optic vesicles and carefully remove the ectoderm. Finally, separate the optic vesicles from the brain with the forceps to dissect an embryo older than stage 25. First, remove the ventral portion of the embryo with forceps in the anesthetization plate.
For better contrast, transfer the embryo to a 60 millimeter plastic Petri dish containing 10 milliliters, 0.1 XMMR and 100 microliters of 1%Nile blue sulfate for two to three minutes to stain the ectoderm. Then transfer it to the dissection plate. Then holding the embryo in place carefully remove the retina or optic vesicle that lies directly below the overlaying ectoderm.
Once the optic vesicle or retina has been dissected, carefully transfer it to the X explan rinse plate with a P 100 micro pipette using an aerosol resistant tip. Be careful not to allow it to touch any air liquid boundaries. Then allow the explan to sit for 30 seconds.
Next, transfer 80 microliters of 0.1%trypsin in CMF from the explan dissociation plate to the explan dissociation tube. After that, transfer the retina or optic vesicle from the explan rinse plate to the explan dissociation tube. Avoiding transfer of the explan rinse solution.
Then allow the tissue to dissociate in the explan dissociation tube for one hour at room temperature. If multiple images of the cells are to be taken throughout the experiment, attach a grid to the underside of the culture plate with small drops of super glue. This will allow images of identical cells in identical orientations to be taken at different points in the procedure to plate the cells.
First, slowly remove 40 microliters of the solution from the explan dissociation tube and discard. Then use the P 100 with an aerosol resistant tip to slowly transfer the cells to the culture plate. When aspirating the cells onto the plate, pipette slowly and keep the cells in a small area in the center of the plate.
This figure shows an example of the calcium activity of a single cell over one hour of imaging. These figures provide the representative results of calcium imaging experiments in embryonic retinal cells as analyzed at different developmental stages. Briefly, the results show that calcium is developmentally regulated with spiking peaks at stage 35 in terms of correlating calcium activity with cells positive for a specific probe.
In fish experiments, while there were no statistically significant differences, there was a trend for cells positive for a GABAergic marker to display higher levels of calcium spiking activity than cells positive for a glutamatergic marker or for PT F1 A.A gene encoding, a transcription factor correlated with promoting the GABAergic phenotype Once mastered, this technique can be completed in about three hours and 30 minutes for one plate if performed properly. Following this procedure, other methods such as cell culture and C two hybridization or electrophysiological recordings can be used to answer additional questions such as which cells express specific phenotypic markers or to analyze electrophysiological recordings. After watching this video, you should have a good understanding of how to dissect retinal tissue, dissociate retinal tissue, and plate retinal cells.
