The overall goal of this procedure is to quantitatively measure relative retinoic acid levels in tissue preparations, such as E8.5 mouse embryos or adult neurosphere cultures, using a cell-based reporter assay. While the RA signaling pathway is well characterized, direct histological measurement of RA is technically unfeasible. This assay can help answer key questions in how genetic mutations can affect RA levels.
The main advantage of this technique is that it is highly sensitive and versatile, with the ability to measure a wide range of RA concentrations in a variety of tissue samples. Though this method can provide direct insight into determining RA levels in various tissue samples, it can also be applied to other studies, such as determining the presence or absence of RA metabolic enzymes by monitoring changes in RA levels. Before starting any experiments, test the response of F9 RARE-LacZ cells to RA.Add one nanomolar of all-trans RA to F9 RARE-LacZ cells plated in a 96-well plate.
Then, incubate the plate, and culture at 37 degrees Celsius overnight. The next day, perform LacZ staining and check the cells for a blue precipitate. If the color is not uniform throughout the cells, proceed to subclone the cells.
Split the selected cultures into 10 centimeter dishes at 100, 000 cells per 10 milliliters. After two days of culturing, prepare a 24-well plate with 500 microliters of 0.2%gelatin per well, and let the plate sit at room temperature for 30 minutes. Then, remove the gelatin, and rinse the wells twice with 500 microliters of distilled water.
Now, add 500 microliters of F9 RARE-LacZ cell culture medium to the wells. Next, pick colonies of F9 cells using a sterile P10 pipette tip and transfer each into one well. After three or four days of culturing, split the colonies into two gelatin-coated 24-well plates, one for testing and the other for expanding.
After two more days of culture, add one nanomolar of RA to each well of one 24-well plate and culture it overnight. The next day, perform a LacZ stain to test for high responders and expand the corresponding culture. One day prior to harvesting the E8.5 embryos or dissociating the neurospheres, coat a 96-well plate with 100 microliters of 0.2%gelatin per well, and let the plate sit for 30 minutes.
Then, aspirate out the gelatin, and rinse the wells twice with 200 microliters of distilled water. Next, trypsinize the selected F9 RARE-LacZ cells maintained in 10 centimeter dishes. Then, plate 100, 000 cells into each well of the 96-well plate using culture medium without G418.
Load enough wells to co-culture each embryo, or prepare three wells per genotype if working with neurospheres. Also prepare 24 wells for the standard curve. Then, incubate the plate for 24 hours.
For embryonic cell co-culturing, begin dissection of E8.5 embryos. Next, load a new 1.5 milliliter tube with 10 microliters of trypsin, and using widened bore P20 pipette tip, transfer the whole embryo into the tube, and place the tube on ice. After all the embryos have been isolated, incubate all the tubes at 37 degrees Celsius for five minutes to dissociate the tissues.
After five minutes, gently triturate each tubes'contents using a P20 pipette tip, and transfer 10 microliters to a well of the prepared 96-well plate with F9 RARE-LacZ cells. Culture the plate overnight. For neurosphere co-cultures, begin with transferring a neurosphere culture to 15 milliliter tubes.
Spin the tubes at 200 g's for 10 minutes, and remove all but one milliliter of the supernatant. Then, triturate the contents with a P1000 pipette. Now, use a small volume to determine the density of the dissociated cells.
Then, plate 100, 000 dissociated neurosphere cells over the F9 RARE-LacZ cells. Load three wells with each neurosphere culture type. Now, be sure to prepare the wells for the standard curve.
In triplicate, add 100 microliters of all-trans RA solutions prepared by serial dilution at seven different concentrations in F9 RARE-LacZ culture medium. Include negative controls of untreated cells. Finally, culture the fully-loaded 96-well plate overnight.
The next day, remove the medium and fix the the cultures using 100 microliters of 2.5%glutaraldehyde per well. Let the plate incubate at room temperature for 15 minutes. Later, remove the fixative and wash the wells twice with 200 microliters of PBS for 10 minutes per wash.
Next, wash the wells three times with 200 microliters of LacZ wash solution for 10 minutes per wash. During the third wash, prepare the X-gal staining solution in a 15 milliliter tube wrapped in foil. Also, prepare a humidified chamber for the 96-well plate.
Now, add 200 microliters of the X-gal staining solution to each well. Place the plate inside the humidified chamber, and incubate at 37 degrees Celsius. After the incubation, remove the LacZ staining solution and replace it with 200 microliters of PBS.
Then, measure the absorbance at 610 nanometers, and image the plate. The F9 RARE-LacZ reporter cell line is composed of adherent embryonic carcinoma cells that respond quantitatively to RA with a proportional induction of beta-galactosidase. They also have an epithelial morphology.
Despite culturing with G418 selection, the response of these cells to RA will weaken with several passages. Thus, periodic subcloning is necessary to ensure strong and uniform responders to RA.E8.5 embryos homozygous for the vl mutation in Gpr161 are expected to show decreased embryonic RA signaling. Using this described co-culture assay of dissociated embryos with F9 RARE-LacZ cells, this expectation was confirmed.
Co-culture of wild-type adult-derived neurospheres with F9 RARE-LacZ cells was also used to test for RA in neurosphere cultures. The increased staining, compared to stage E8.5 cells, may be due to the homogeneity of neurosphere cultures. By generating a standard curve for RA levels, the RA level was quantifiable in wild-type neurosphere cultures and in E8.5 embryos.
While attempting this procedure, remember to maintain through periodic subcloning F9 RARE-LacZ cells that respond strongly to RA.Also, be sure to dissociate your sample thoroughly to facilitate homogenous distribution and consistent RA measurement. Following this procedure, the effects of genetic mutations on levels of RA can be investigated, and other methods can then be performed to investigate which components of the RA signaling pathway is directly affected.
