The overall goal of this procedure is to isolate embryonic ventricular endothelial cells. This is accomplished by first retrieving an embryo for dissection. Next, the heart is excised.
The atri removed and the minced ventricles are transferred into a collagen solution. The single cell suspension is then incubated with an antibody that recognizes the endothelial cells. And finally, a magnet is used to isolate the antibody bound endothelial cells.
Ultimately, results can be obtained that show endothelial chain formation through bright field microscopy. This method can help answer key questions in the cardiovascular development field, such as what factors promote coronary endothelial migration. Though this method can provide insights into endothelial cell behavior, it can also be applied to other cell types such as myocytes and fibroblasts On the day prior to performing the dissection working in a tissue culture hood, combine the antibody of choice with the appropriate dyna beads following the manufacturer's instructions for the rat anti CD 31 antibody used here.
Three microliters of antibody is added to 25 microliters of beads for a final concentration of 1.5 micrograms in 10 to the seven beads incubate beads in antibody overnight at four degrees Celsius to prepare collagen gels for quick tubule formation of isolated cells in a tissue culture hood on ice. Combined collagen type one sterile water, 10 XM 1 99, and five molar sodium hydroxide. According to the text protocol, pipette 20 microliters of the collagen gel into the wells of a 384 well tissue culture plate.
Place the plate in a 37 degree Celsius tissue culture incubator for 30 minutes. Add 80 microliters of DMM to each well and incubate for 15 minutes at 37 degrees Celsius. Remove the culture medium and repeat the wash two more times after the final rinse, add 80 microliters of 10%FBS DM to each well and incubate overnight at 37 degrees Celsius.
After euthanizing a timed pregnant mouse at the desired embryonic day. Using an approved technique and removing the uterine horn according to the text protocol, place it in a Petri dish with one XPBS and rinse it. Once the embryonic sacks have been exposed via a midline incision at the junction of the placenta and an embryo, cut open an embryonic sack and retrieve the embryo.
Place the embryo in a second Petri dish with cold PBS and return the uterine horn to ice to improve access to the chest wall de capitate the embryo. And if genotyping, cut the tail and save it in a tube on ice. With the embryo on its back cut.
Open the chest wall to visualize the heart and lungs to avoid cutting the heart. Make a vertical cut along the side of the rib cage near a four limb, followed by a horizontal cut across the bottom of the ribs. Then gently pull the chest wall up and out of the way up through approximately embryonic day 13.5.
The chest wall is transparent, aiding in visualization. Using forceps, carefully lift the heart and cut the vessels below. If desired, transfer the heart to a clean dish to free the heart from the lungs, cut above the great vessels.
Next, separate and discard the atria and great vessels from the ventricles. Then using scissors, mince the ventricles into small pieces and place them in approximately 500 microliters of collagenase on ice. Excise and process the ventricles from the remaining embryos, collecting each heart in a separate tube.
Place the minced ventricles at 37 degrees Celsius with rocking for 45 minutes Every 15 minutes, gently remove the samples and pipette up and down to mechanically dissociate the cells. After the incubation, pass the ventricle collagenase solution through a 70 micrometer filter to remove remaining clumps of cells, centrifuge the cells at 400 G for 10 minutes, discard the supernatant and replace with approximately 200 microliters of 10%FBS in DMEM pipette up and down to resuspend the cells, spin the cells again, and then after a second wash and spin. Resus suspend the cells in 50 microliters of 10%FBS in dmm.
Next, prepare the beads by first placing the tube of antibody and beads on a magnet for one minute, aspirate the supernatant. Then remove the tube from the magnet and add approximately 100 microliters of 10%FBS in dmm. Return the tube to the magnet for one minute, then remove the supernatant.
Repeat the wash a total of three times after the final wash. Resuspend the antibody conjugated beads in 10%FBS in DMM to each cell sample, add five microliters of the antibody conjugated beads. Place the bead cell suspensions at four degrees Celsius with rocking for 30 minutes in a laminar flow hood, place the samples on the magnet for one minute, aspirate the supernatant.
Remove the tubes from the magnet and wash with approximately 100 microliters of 10%FPS and dm. Repeat for a total of five washes after the final wash. Suspend the beads in approximately 100 microliters of DMEM.
Place the tubes on the magnet for one minute. Remove the medium, then add 100 to 200 microliters of prewarm, 0.25%trips in EDTA, incubate the samples at 37 degrees Celsius and 5%carbon dioxide for five minutes. Place the tubes on the magnet for two minutes.
Then carefully transfer the cell containing supernatant to fresh tubes and centrifuge them for five minutes at 400 G.Finally, after removing the supernatant Resus, suspend the cells in 80 to 100 microliters of endothelial growth, medium and plate. In a 96 or 384 well treated culture dish, depending on the expected yield, each pair of ventricles from E 14.5 through E 16.5 embryos, typical yields 450 to 600 cells as demonstrated here using an endothelial specific antibody that recognizes CD 31. Endothelial cells were isolated from the ventricles of day E 13.5 embryos.
When grown on an untreated culture dish, these cells remain rounded and form a cobblestone pattern when near confluent. Although the cells will adhere to plates coated in dilute collagen, these cells isolated from E 18.5 ventricles form fewer chains than when coated on a collagen gel. In addition, the cells more rapidly form cell cell interactions and form chains, the branch on the collagen gel versus the collagen coated plate as verification of their identity.
The cells in this figure label positively with the endothelial marker ISO selectin. In addition, as shown here, endothelial cells isolated from E 18.5 ventricles labeled positively with the endothelial markers, PAM and flick one. This figure shows endothelial cells that were isolated from the ventricles of an E 14.5 embryo and labeled with the endothelial specific dye cyto.
16 isolated cells survive up to one weaken culture, but are resistant to ization and are therefore best suited for terminal experiments. Once mastered, this technique can be done in approximately three and a half hours if performed properly. Following this procedure, other assays such as tube forming assays and transwell migration assays can be performed to determine how these isolated cells behave in vitro.
