Explant Coculturing

The cell plating and the incubations of embryonic tissue must be synchronized to give confluent F-9 cells on the day embryonic tissue is ready for explanting. Superconfluent F-9 cultures should be avoided, since the cells pile up and this makes it more difficult to keep the explant attached to the mono-layer of cells.

1. Dissect the tissue explants in a balanced salt solution. If the ectoderm is trypsinized off, for example, or any other enzymes used to separate tissues, it is very important to wash the tissue and neutralize the enzymes with medium with serum prior to explanting, since any remaining trypsin will destroy the monolayer.

2. Immediately before placing the explant on the cell monolayer, remove the growth medium from the F-9 cells and replace with 1 mL serum-free medium. Explants are always cultured in serum-free medium, since serum has retinoids in it.

3. Transfer the explants onto the cell monolayer with a pipet.

4. The dishes should be moved as little as possible to allow the explants to settle and attach to the cell monolayer. If attachment proves to be a problem, minimizing the medium over the cells when first introducing the explant can increase cell contact. After a hour or so, gently add more medium to these cultures to ensure that they do not become dry overnight.

5. Culture at 37°C, in a 5% CO2 atmosphere overnight.

6. Wash the cultures twice very gently (in order not to disturb the F-9 cells) with room temperature PBS. Then fix for 5 min at 4°C with 2% paraformaldehyde, 0.2% glutaraldehyde in PBS.

7. Rinse gently twice more with PBS, then cover with 1 mL of X-gal stain/dish, and incubate at 37°C overnight.

8. Count the number of blue cells around the explant as a percentage of all cells.

Each time an experiment is performed with tissue explants, a series of control RA dilutions need to be run in parallel, which is why one needs about 20 dishes. A typical series would be 10-6-10-11 M in steps of 10-fold dilutions. In the case of F-9 cells with the lacZ gene, the results are determined by taking a field of view down an inverted microscope and counting the number of blue cells as a percentage of the total cells. The plate is then moved to another field and the counts repeated. The result of such an experiment is shown in Fig. 1A. When the explants are counted, then an approximation of the amount of retin-oid present can be made by comparing % blue cells with the control curve.

Several explants of the same tissue type can be placed in one Petri dish. Usually, the explants remain in place all through the P-gal staining process, but if they do not, that is rarely a problem because a ring of blue cells marks the place where they were and counts can still be performed. Counts are made of the percent blue cells within a fixed distance from the explant.

Obviously, 50 cell diameters away from the explant, none of the cells are blue, whereas the majority of those directly touching the explant will probably be blue, so a compromise in distance much be reached and adhered to for all explants. A typical result for chick limb buds cut into two halves is shown in Fig. 1B. By comparison with the standards in Fig. 1A it is possible to suggest that the posterior halves of chick limb buds contain in the region of 10-9 M RA and that the anterior halves contain considerably less. This result fits well with the original HPLC data of Thaller and Eichele (3).

Finally, it is important to remember that this assay does not measure tRA alone because several retinoids are equally efficient at activating the RARs,

Fig. 1. (A) Calibration curve generated by counting the number of F-9 reporter cells that have turned blue after treatment with increasing concentrations of tRA. A straight line is usually obtained. From these data, an approximation of the concentration of RA present in explanted tissues can be made from the % blue cells recorded in the explant dishes. Bars mark standard deviations. (B) Example of data obtained after explanting anterior (ant) and posterior (post) half chick limb buds at two different stages of development, stage 20 and stage 23. It is clear that posterior halves contain more RA than anterior halves. A value of 20% blue cells from stage 20 posterior halves gives a concentration of close to 10-9 M RA from the standard curve in A.

Fig. 1. (A) Calibration curve generated by counting the number of F-9 reporter cells that have turned blue after treatment with increasing concentrations of tRA. A straight line is usually obtained. From these data, an approximation of the concentration of RA present in explanted tissues can be made from the % blue cells recorded in the explant dishes. Bars mark standard deviations. (B) Example of data obtained after explanting anterior (ant) and posterior (post) half chick limb buds at two different stages of development, stage 20 and stage 23. It is clear that posterior halves contain more RA than anterior halves. A value of 20% blue cells from stage 20 posterior halves gives a concentration of close to 10-9 M RA from the standard curve in A.

including didehydroretinoic acid and 9-cis. Therefore, what one measures here is the combined concentration of the retinoic acids.

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