Multicellular tissues complex, this protocol reconstitutes cellular conduct in a simplified manner. One thing is this technique to study the direct relationships between cellular conductor protons and cellular response. This protocol uses chemically functional polystyrene beads to reconstitute the cellular conductor protons.
The beads can be cut with any protons of interest, to test their effects on cellular response. We have used this method in C-Elegance on mouse embryos. Therefore, this protocol can be upright to the studies of wide organisms.
As embryos are fragile after high full chlorite treatment, significant practice is required before one can successfully isolate blastomere. Visual demonstration will provide a greater understanding of how one should handle capillary during blastomere-isolation methods. To begin, weigh 10 milli-grams of carboxylate modified polystyrene beads in a 1.5 milli-liter micro centrifuge tube.
Add one milli-liter of MES buffer into the tube to wash. Vortex the tube to mix the beads. Spin the tube for 60 seconds at 2000XG via bench-top centrifuge.
Dicard the supernatant by carefully pipetting out the buffer. Wash the beads again with one milli-liter of MES buffer. After the wash, add one milli-liter of MES buffer containing 10 milli-grams of EDAC into the tube, to activate the surface carboxyl groups.
Vortex the tube to mix the beads. Rotate and incubate the tube for 15 minutes at room temperature. Spin down the beads for 60 seconds at 2000XG.
Discard the supernatant by carefully pippetting out the buffer, and wash with one milli-liter of PBS. Vortex the tube to mix the beads and repeat the PBS wash one more time. Prepare one milli-liter of one, 10, 100, and one-thousandfold dilution series of Rhodamine Red-X from the 0.65 milli-liter Rhodamine Red-X stock solution.
Pippette 20 micro-liters of beads into each serial dilution tube. Rotate and incubate the tubes for five minutes at room temperature. Wash the beads twice with one milli-liter of PBS.
After wash, add one milli-liter of PBS into the tubes and store them at four degrees Celsius for up to six weeks. Check the fluorescence intensity of the beads under a microscope used for live imaging. Hold each end of a micro-capillary at a capacity of 10 micro-liters with right and left hand.
Pull the micro-capillary towards both ends to apply tension and bring the center of the capillary over a burner to make two hand-pulled capillaries. Under the dissecting microscope, trim the tips of the hand-pulled capillaries with forceps. Make the two tip opening sizes approximately two times, and one times the short access length of C-Elegance Embryos for the embryo transfer and eggshell removal respectively.
Attach the pulled capillary into a mouth-pippetting apparatus. Pippette 45 micro-liters of egg-salts solution onto a well of a multi-welled slide. Place five to 10 adult C-Elegance onto the well.
To obtain early C-Elegance Embryos cut adults into pieces by positioning two needles to the right and left of C-Elegance's body, and sliding the needles past each other. Pippette 45 micro-liters of hypochlorite solution onto a well next to the well containing egg-salt solution and pippette 45 micro-liters of Shelton's Growth Medium onto the subsequent three wells. Transfer one cell stage and early two cell stage embryos into the hypochlorite solution by the mouth pippette with the larger sized opening and wait for 40 to 55 seconds.
Wash the embryos by transferring them into the next three wells with Shelton's Growth Medium. With one to two seconds in each of the first two wells. Using the hand-drawn capillary with the smaller sized opening carefully repeat pippetting the embryo for eggshell removal.
After that, continuously pippette with the hand-drawn capillary to separate the two-cell stage embryonic blastomeres. After eggshell removal, minimize the force in pippetting the embryos up and down to prevent burst. Prepare an imaging chamber by placing a coverslip onto a coverslip holder and tape the edges of the coverslip to stabalize it.
Flip the coverslip holder, and draw a circle on the coverslip with a hydrophobic pen. Add Shelton's Growth Medium within the circle drawn by the hydrophobic marker, and transfer the isolated blastomere to the imaging chamber within the circle. Now, dispense a small volume of the chemically functionalized beads onto the coverslip using the hand-drawn capillary with the larger opening.
Control the position of the beads by blowing into the hand-drawn capillary until the beads attach to the isolated blastomere. Mount the coverslip to avoid evaporation of medium and perform live imaging using an inverted microscope. In this study, the fluorescent signal from the bead treated with 0.005 micro-grams per milli-liter Rhodamine Red-X Succinimidyl Ester for the transgenic strain expressing GFP miosin-2 and M-cherihistone was too weak.
On the other hand, the fluorescent signal from the beads treated with five micro-grams per milli-liter Rhodamine Red-X Succinimidyl Ester was too strong. The concentration of Rhodamine Red-X Succinimidyl Ester at 0.5 micro-grams per milli-liter was optimal for this particular transgenic strain. During the live imaging a plane was identified where two centrosomes were vertically aligned.
The plane with plus or minus 90 degree angle of this plane is spindle plane. The angle of spindle orientation relative to the cell/bead contact interface after cytokinesis shows that both daughter cells were attached to the bead in a line that passes both contact sights was used as a contact orientation. One needs to learn how embryos and eggshell react to certain external forces applied by the mouth pippette.
Theoretically, any cellular response can be studied, for example, sulfate specification can be studied by culturing cells and contact with adhesive beads over longer terms. This technique has been used to understand contact dependent cell division orientation mechanism. PTF filter was used to prevent inhalation of fumes of hypochlorite solution via mouth pippette.
