Fly crosses and virgin collection:
1| Cross UAS-mCD8-GFP females in bottles to either slbo-Gal4/CyO (for marking border cells and centripetal cells, see Figure 1B-C), or c522 (for marking border cells only).
2| Collect slbo-Gal4/UAS-mCD8-GFP and UAS-mCD8-GFP /+; c522/+ virgins and age at 18°C for 2-7 days, then mate with males of the same genotype and fatten (by adding yeast paste) overnight at 18°C and 25°C respectively before dissection.
3| Cross w1118 females to UAS-mCD8-GFP / CyO; tub-Gal4/TM6B. tub-Gal4 is expressed in all follicle cells.
4| Collect UAS-mCD8-GFP /+; tub-Gal4/+ virgins and age at 18°C for 2-7 days, then mate with males of the same genotype and fatten overnight at 18°C (for the comparison with slbo-Gal4/UAS-mCD8-GFP) or 25°C (for the comparison with UAS-mCD8-GFP /+;c522/+ ) before dissection.
1| Dissect ovaries, as diagrammed in Figure 1D-E, in Grace’s medium with 10% normal goat serum (final pH should be adjusted to 6.9-7.0).
2| After dissecting about 10 ovaries, transfer them to a tube on ice. Keep the total time for dissection to less than two hours. As shown in Figure 1B-C, the mCD8-GFP+ population represents a small fraction of all the cells in the dissected tissue. Recently we have found that egg chambers survive and border cells migrate at 25°C, but when put at 4°C, they retract protrusions (M.M. and D.J.M., unpublished). We do not know if gene expression patterns are altered as a function of temperature but it may be just as good, or even better, to keep the egg chambers at room temperature during the dissection, rather than putting them on ice. This needs to be determined empirically.
1| Reconstitute elastase in cell dissociation buffer at a concentration of 4mg/ml. This solution can be stored in aliquots at –20°C, but multiple freeze-thaws should be avoided. Prior to the experimental sample, the elastase solution should be tested (see Critical step).
2| Collect about 100 ovary pairs in a 1.5ml centrifuge tube, wash with cell dissociation buffer three times and digest with 1ml of elastase solution.
3| During digestion, incubate tubes at room temperature and either agitate by inverting by hand, or place the tubes on a stir-plate standing on their caps, and mix with a micro-stir bar that fits into the tube cap. Agitation is important to dissociate the tissue fully.
4| Add 0.5 ml of supplemented Grace’s medium, and transfer the supernatant to a new tube (2 times).
5| Centrifuge dissociated cells at 1000g for 5 minutes at 4ºC.
6| Resuspend pelleted cells in supplemented Grace’s media or PBS buffer + 0.5% BSA for sorting.
Filter the dissociated cells using Filcon 50µm filters to remove clumps and then subject to magnetic cell purification with magnetic beads coupled with anti-mouse CD8 antibody (MACS, Miltenyi Biotec, "http://www.miltenyibiotec.com":http://www.miltenyibiotec.com) according to the manufacturer’s manual. Briefly:
1| Centrifuge filtered cells at 1000g for 5 minutes at 4ºC, resuspend in 90 μl of buffer per 10-20 ovary pairs, then add 10 μl of MACS CD8a microbeads, and incubate tubes in a refrigerator (8-12ºC) for 15 minutes.
2| Wash the cells bound to the beads in 1ml of buffer, spin down, and resuspend in 250 µl of buffer. Meanwhile, place a positive selection column in the magnetic field of the MACS separator and equilibrate it with 500 μl filtered buffer.
3| Apply 500 μl of cell suspension in buffer to the pre-filter over the column. After the cell solution passes through (keep the flow-through in two tubes as the CD8- control), wash the column three times with 500 μl buffer.
4| Remove the column from the MACS separator, place it over a 5ml collection tube, and add 1 ml of buffer. To collect the CD8 positive cell fraction, flush out the column with the supplied plunger.
5| For RNA preparation, spin down the cells, take off the supernatant, and proceed to the next section. Alternatively, the cell pellets can be frozen at this point for later RNA isolation. An overview of the ovary dissection, cell dissociation and cell purification procedure is shown schematically in Figure 1A. A sample of purified cells is shown in Figure 1F-I.
cRNA probe labeling and hybridization:
1| Prepare total RNA from purified cells as directed in the Qiagen RNeasy protocol or equivalent protocol. As a starting point, use 80 μl RLT buffer for lysis of the positive cell fraction, and 160 μl buffer for the negative cells. For each experiment (genotype), isolate RNA independently in triplicate.
2| Large scale or small scale experiments can be carried out, depending on the amount of starting material. For our initial large scale experiments, 5 μg of each RNA from slbo-Gal4/UAS-mCD8-GFP and UAS-mCD8-GFP / +; tub-Gal4/+ flies was used to make cRNA probes following Affymetrix probe labeling instruction ( "http://www.affymetrix.com/index.affx":http://www.affymetrix.com/index.affx). However, we also had success with small scale experiments, which require much less starting tissue. 30 ovary pairs are sufficient to yield more than 100ng of RNA. For this strategy, isolate 100ng of each RNA from UAS-mCD8-GFP /+; c522/+ and slbo-Gal4/UAS-mCD8-GFP and UAS-mCD8-GFP / +; tub-Gal4/+ flies to make cRNA probes by following the protocol Small Sample Target Labeling Assay Version II ( "http://www.affymetrix.com/index.affx":http://www.affymetrix.com/index.affx). Briefly, synthesize double-stranded cDNA using Invitrogen Superscript II, then amplify the cRNA pool with the Ambion MEGAScript T7 kit and a second round of cDNA synthesis. Amplify and label the cRNA using the ENZO BioArray High Yield RNA Transcript labeling kit. Hybridize cRNA probes to Affymetrix Drosophila Genome Array as directed, then wash and proceed to signal detection as suggested by Affymetrix. Each experiment should be repeated three times using independent biological samples.
Variations on the protocol
This protocol can be used to purify any population of cells for which there is a specific GAL4 driver. In addition, by crossing the GAL4 driver of interest and UAS-mCD8-GFP in mutant backgrounds, the same population of cells can be purified from wild-type and mutant strains. For example, to identify transcriptional targets of the Slow Border Cells (SLBO) transcription factor, one can isolate border cells from slbo-Gal4, slboe7b / UAS-mCD8-GFP, slboLy6 flies to compare these loss-of-function mutants to wild type. As the slbo-Gal4 driver also activates expression in the centripetal cells, a different driver may be needed. We used c522, slboe7b / UAS-mCD8-GFP, slboLy6 to identify SLBO targets that might be specific to the border cells. As we expected, this method uncovered some known and many new potential target genes for SLBO. In a similar type of experiment, we sought to identify targets of the JAK/STAT signaling pathway. In this case, we used slbo-Gal4, UAS-mCD8-GFP / UAS-UPD because high levels of the activating protein Unpaired (UPD) result in hyper-activation of STAT target genes.