This protocol allows for the seamless isolation, culturing, and interrogation of transcriptional responses in a specific cell lineage from C.elegans. The main advantage of this protocol is the high level of adaptability to different life stages of worms or specific cell types to be isolated. Demonstrating the procedure will be myself and Nataliya Zahayko, a technician from our laboratory.
After collecting the animals, centrifuge them at 1600 times g for five minutes. Remove all of the supernatant and resuspend the worms in one milliliter of M9 media. Transfer the suspension to a 1.5-milliliter microcentrifuge tube.
Centrifuge at 1600 times g for five minutes to pellet the worms. Then add 200 microliters of SDS-DTT buffer and incubate at room temperature for five minutes. The worms should now appear to be wrinkled along the body if viewed under a light microscope.
Next, add 800 microliters of ice-cold isolation buffer and mix by gently flicking the tube. Centrifuge at 13, 000 times g and at four degrees Celsius for one minute to pellet the worms. Remove the supernatant and wash with one milliliter of isolation buffer.
Repeat the centrifugation and wash process a total of five times, making sure to carefully remove the isolation buffer each time. After this, add 100 microliters of protease mixture from Streptomyces griseus, dissolved in isolation buffer, to the pellet. Incubate at room temperature for 10 to 15 minutes, making sure to apply mechanical disruption by pipetting up and down 60 to 70 times with a 200-microliter micropipette tip against the bottom of the tube.
To determine the stage of digestion, remove one to five microliters of the digestion mixture, drop it onto a glass slide, and inspect it using a tissue culture microscope. After five to seven minutes, worm fragments should have a visibly reduced cuticle and a slurry of cells will be readily visible. Halt the reaction by adding 900 microliters of commercially-available Leibovitz's L-15 medium, supplemented with 10%FBS and penicillin-streptomycin.
Centrifuge at 10, 000 times g and at four degrees Celsius for five minutes to pellet isolated fragments and cells. Wash the pelleted cells twice more with cold L-15 supplemented media using one milliliter of media per wash. Resuspend the pelleted cells in one milliliter of L-15 supplemented media, and leave on ice for 30 minutes.
Then take the top layer, which is approximately 700 to 800 microliters, and transfer it to a microcentrifuge tube. Use an automated cell counter or hemacytometer to measure the cell density of 10 to 25 microliters of isolated cells. Centrifuge cells from the isolated cell suspension at 10, 000 times g and at four degrees Celsius for five minutes.
Discard the supernatant and resuspend the pelleted cells in L-15 supplemented medium to a cell density of no more than six million cells per milliliter to avoid overloading the flow cytometer. Then, sort cells by GFP-positive expression using a flow cytometer capable of sorting samples. GFP-negative cells can be used as a control for non-cell-type-specific analysis.
After this, plate the isolated cells into the wells of a sterile six-well plate, place the plate into a plastic container, add a damp wipe or soft tissue paper to add moisture to the cells, and incubate at 20 degrees Celsius. Centrifuge cells from the isolated cell suspension in a 1.5-milliliter RNase-free microcentrifuge tube at 10, 000 times g and at four degrees Celsius for five minutes. Using a micropipette, remove the supernatant and add 100 microliters of M9 medium to wash away the L-15 supplemented medium.
Next, centrifuge the cells at 10, 000 times g and at four degrees Celsius for five minutes and remove the supernatant. Repeat this wash and centrifugation process three times total to ensure that all the media is removed, making sure to carefully remove the supernatant each time. Then, add one milliliter of phenol and guanidine isothiocyanate solution to the cells and pipette the suspension up and down carefully.
Incubate the mixture at room temperature for five minutes. After this, add 250 microliters of chloroform and gently invert the tube 15 to 20 times. Incubate at room temperature for five minutes.
Centrifuge at 10, 000 times g and at 25 degrees Celsius for five minutes. Use a micropipette to carefully remove as much of the top aqueous layer as possible, making sure to not disturb the bottom organic layers. Transfer the bottom layer to a new 1.5 milliliter RNase-free microcentrifuge tube.
To the new tube, add 500 microliters of isopropanol and mix by inverting the tube. Incubate this solution at room temperature for five minutes. Then centrifuge the tube at 14, 000 times g and at 25 degrees Celsius for 20 minutes.
Place the samples on ice and use a micropipette to remove as much isopropanol as possible. Gently add one milliliter of 70%ethanol in RNase-free double-distilled water to the tube by applying it to the side of the tube. Centrifuge at 10, 000 times g and at 25 degrees Celsius for five minutes.
Remove most of the ethanol and let the tube air-dry on ice for three to five minutes. After this, add 15 to 20 microliters of RNase-free double-distilled water. Use a spectrophotometer at 260 nanometers to measure the RNA concentration and purity.
In this study, unc-17:GFP-positive cholinergic neurons are extracted and isolated from the roundworm C.elegans for subsequent ex vivo studies. The unc-17 gene is expressed throughout the C.elegans body, and codes for a vesicular acetylcholine transmembrane transporter. The expression of this gene can be observed in head, midbody, and tail neurons, and neuron projections.
Representative fluorescence micrographs of unc-17:GFP-positive neurons after isolation, as well as a respective negative control from unmodified N2 wild type animals, are shown here. Isolated cells can stay alive up to three days when supplemented with Leibovitz's L-15 medium and 10%FBS. Representative data of qPCR from GFP-expressing cholinergic neurons, as well as GFP-expressing muscle cells, is shown here.
After successfully sorting cells, RNA is isolated via a phenol and guanidine isothiocyanate method, after which cDNA was produced using a synthesis kit. Expression of cholinergic neuron-specific genes, tph-1, a tryptophan hydroxylase, and snb-1, a synaptic vesicle transporter, is elevated in unc-17:GFP isolated cells. However, this expression is not present in myo-3:GFP isolated cells.
The inverse is true with the expression of the muscle-specific myosin-heavy chain transcript of myo-2, as the expression of this gene is limited to isolated muscle cells, but not the isolated cholinergic cells. It is important to incubate the worms fully. However, extended incubation of the worm cuticle can result in worm death or additional stress to the animal.
Following the isolation, the cells can either be cultured for a period of time, or used for RNA isolation and qRT-PCR analysis. Cultured neurons can be further analyzed by patch clamp electrophoresis measurements. This technique might facilitate ex vivo analysis of specific groups of cells, thereby helping to improve our understanding of cell-specific responses in the context of multicellular organisms at various stages of its lifecycle.
As chemicals such as SDS and DTT are used during this experiment, general lab safety procedures should be followed while making and using buffers.
