This method can help address key questions in biology, such as how diverse cell types are generated and organized into higher order structures with specific functions. The main advantage of this technique is that it facilitates efficient purification of cells present at a low abundance in the Drosophila visual system for transcriptomic and genomic analysis. On Tuesday of week six of the protocol, 45 minutes before the dissections, retrieve one vial of papain powder from four degrees Celsius and incubate it at room temperature.
Next, set aside Complete Schneider's Medium, or CSM, in an Eppendorf tube at room temperature to add to the papain powder later. Five to ten minutes before the dissections, use a syringe to add the room temperature CSM to the vial of papain directly through the cap to a concentration of 100 units per milliliter. To mix, first invert the vial to capture any powder stuck on the inside of the cap and then pipette the contents up and down.
After mixing the reagents, bring the water up to 37 degrees Celsius in a beaker in a water bath. Then place the vial in the beaker for 30 minutes to activate the papain and make sure that the vial is not floating. While the papain is incubating, retrieve an aliquot of proteolytic enzyme blend from the freezer and incubate it at room temperature.
After 30 minutes of activation, incubate the papain at room temperature. On Tuesday of week six, dissect the staged pupil brains in cold CSM with clean forceps. Dissect as many brains as possible in 10 minutes and pool the brains in a fresh drop of cold CSM.
Cut off the optic lobes by pinching at the junction of the optic lobe and the central brain. Next, add the lobes to the bottom of a microtube containing 500 microliters of 1x RS.One microtube for the experimental tissue and one for the negative control tissue. Keep the tubes on ice.
Dissect as many lobes as possible in one hour. Pool the dissected optic lobes in a single microtube to eliminate tissue loss during transfer between microtubes. Carefully remove the 1x RS from the microtube with the dissected optic lobes using a P200 pipette, leaving enough solution to cover the lobes.
Carefully add 500 microliters of 1x RS to the side of the tube. Let the lobes settle to the bottom of the tube, then pipette up 200 microliters. Expel the mix, observe the lobes moving and allow the lobes to settle again.
For the two samples, aliquot 600 microliters of activated, room temperature papain into a microtube. Next, create the dissociation solution. Add 4.2 microliters of room temperature proteolytic enzyme blend into 600 microliters of papain solution to obtain a final concentration of 0.18 WU per milliliter and mix the solution by pipetting it up and down.
Then carefully remove the 1x RS from each sample and add 300 microliters of dissociation solution to the lobes. Incubate the samples at 25 degrees Celsius. 1, 000 RPM for 15 minutes in a microtube ThermoMixer.
At the 5 and 10 minute time points, stop the ThermoMixer and let the lobes sink to the bottom of the microtube. Pipette up 200 microliters of the solution and mix. After the 15 minute incubation, wait for the lobes to settle to the bottom and carefully remove the dissociation solution from the lobes without disturbing the lobes.
Wash the lobes two times with 1x RS.Carefully add 500 microliters of 1x RS without disturbing the lobes. Then carefully pipette up 200 microliters and gently expel the solution. Allow the lobes to sink to the bottom and repeat.
Next, wash each sample two times with 500 microliters of CSM. Carefully remove the CSM and add another 200 microliters of CSM to the tube. Using a P200 pipette, disrupt the tissue by pipetting up and down without foaming until the solution is homogenous.
Using a P200 pipette, filter the cell suspension through a 30 micrometer mesh cap into a 5 milliliter fax tube on ice. And make sure the entire suspension goes through. Add 500 microliters of CSM to rinse the dissociation microtube.
And filter the solution into the fax tube. Finally, carefully take off the cap of the fax tube. Collect the solution underneath the mesh filter with a P200 and add it to the rest of the suspension in the fax tube.
Confocal microscopy of immuno stained lobes with antibodies specific against DsRed and GFP as well as the 24B10 antibody as a reference for the Lamina and Medulla neuropils confirmed that L3 is the only cell type labeled by both DsRed and GFP in the optic lobe. Facts data from 100, 000 events were recorded, of which 29.9%of all the events are potential singlets. After doublets in cells with different sizes were gated out based on granularity and size, the remaining single cells, L3 neurons, appeared as tight clusters in P1, which was well-separated from the background cells.
Following this procedure, other methods like RNA-seq or ATAC-seq can be performed to address biological questions through the analysis of gene expression or chromatin organization, respectively. This technique considerably advances the ability of researchers to explore the genetic mechanisms underlying the development and function of complex tissues using the Drosophila visual system as a model.
