Employing Reporter Cells in a Co-Culture to Study Retinoic Acid Production by Mouse Embryonic Cells

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines

1. Solution and Media Preparation

1. Prepare stock and buffer solutions as per Table 1. Prepare working solutions as per Table 2.

2. Culture and Maintenance of F9 RARE-LacZ Cells

1. Thawing Frozen Cells​
   1. Prepare F9 RA response element (RARE)-LacZ cell culture medium as detailed in Table 2.
   2. Coat 10 cm culture dishes (cell culture treated) with 10 ml of 0.2% gelatin (refer to Table 2) for 30 min at RT. Wash off excess gelatin with two rinses of 10 ml distilled water. Immediately add 9 ml of F9 RARE-LacZ cell culture medium into the flask to prevent the gelatin from drying.
   3. Thaw a vial of F9 RARE-LacZ cells in a 37 °C water bath for 2 – 3 min. Plate cells (around 1 x 10⁶ cells/ml) into the coated dish containing 9 ml of culture medium. Culture at 37 °C, 5% CO₂. Change the medium the next day.
2. Maintenance of F9 RARE-LacZ Cells
   1. Maintain a regular passage every 2 – 3 days at a ratio of 1:10.
      Note: Do not let cells grow into confluency as this will result in their differentiation.
   2. Split the cultures at a confluency of 80-90%. Remove the media and wash the cells with 10 ml of 1x phosphate-buffered saline (PBS). Remove the PBS and add 1 ml of trypsin, incubate for 5 min. Add 9 ml of culture medium, pipette up and down and split cells 1:10 for passage.
3. Freezing F9 RARE-LacZ Cells
   1. Trypsinize a 10 cm dish containing F9 RARE-LacZ cells at 80 – 90% confluence as detailed in step 2.2.2. Transfer the dissociated cells into a 15 ml centrifuge tube and spin at 200 x g for 5 min.
   2. Remove supernatant and resuspend cell pellet in 10 ml of F9 RARE-LacZ freezing medium (see Table 2). Aliquot 1 ml into labeled cryovials and transfer vials into freezing containers. Place freezing container in -80 °C freezer overnight (O/N) and transfer vials to liquid nitrogen tanks the next day.

3. Dissection of Embryonic Day 8.5 (E8.5) Embryos

1. Mating Cage Set-up and Plug Check​
   1. Set-up a mating cage containing one mating pair. The morning of the next day, check for the presence of a vaginal plug and designate the day a plug is found as day 0.5. At day E8.5, harvest embryos.
2. Dissection of Embryos
   1. Sacrifice the dam through cervical dislocation and spray the abdominal area with 70% ethanol (see Table 2). Make a transverse cut on the skin above the abdomen using surgical scissors and pull the two flaps apart (anterior and posterior) to expose the abdominal area. Make a similar cut on the peritoneum to expose the abdomen.
   2. Locate the uteri and release uteri from the oviducts and mesometrium using a pair of scissors. Place the uteri in a 6 cm dish (non-cell culture treated) with 10 ml 1x PBS to wash off excess fat and blood. Transfer to another 6 cm dish containing fresh 10 ml 1x PBS.
   3. Separate one uterus containing one embryo by cutting it off using surgical scissors. Remove the uterus and decidua and release the yolk sac containing the embryo using two pairs of no. 5 forceps.
   4. Separate the yolk sac from the embryo and transfer the yolk sac to a 1.5 ml microcentrifuge tube for genomic DNA extraction to be used for genotyping. Use a P20 pipette with a wide-bore tip, transfer the whole embryo into a 1.5 ml microcentrifuge tube containing 10 µl of trypsin. Place the tube on ice.
   5. Repeat steps 3.2.3 – 3.2.4 until all embryos have been dissected.
3. Embryo Dissociation
   1. Incubate the 1.5 ml tubes containing embryos in trypsin at 37 °C for 5 min to facilitate enzymatic dissociation.
      1.  Triturate gently using a P20 pipettor for mechanical dissociation of the embryo, and plate the whole 10 µl volume containing one dissociated embryo over one well of F9 RARE-LacZ cells in a 96-well plate (see step 4.1 for plating F9 RARE-LacZ cells in a 96-well plate). Culture O/N at 37 °C and 5% CO₂.

4. RA Assay of E8.5 Embryos

1. Plating F9 RARE-LacZ in 96-well plate​
   1. One day prior to harvesting E8.5 embryos or dissociation of neurospheres, coat a 96-well plate with 100 µl of 0.2% gelatin per well for 30 min at RT. Aspirate out the gelatin and rinse twice with 200 µl of distilled water.
   2. Trypsinize F9 RARE-LacZ cells maintained in 10 cm dishes as detailed in step 2.2.2. Plate 1 x 10⁵ cells/well of F9-RARE LacZ cells, but this time using culture medium without G418.
   3. Prepare enough wells for co-culture of embryos (~ 12 – 20, depending on litter size), neurospheres (3 wells/genotype) as well as for a standard curve in triplicate (27 wells).
2. Co-culture of F9 RARE-LacZ and E8.5 Embryos
   1. Harvest embryos as detailed in section 3.2 and plate on top of F9 RARE-LacZ in the 96-well plate from section 4.1. Culture O/N at 37 °C and 5% CO₂.

Table 1. Buffers and Solution Compositions.

Table 2. Working Buffers and Solution Compositions.