The overall goal of this procedure is to isolate lipid droplets from tissue. This is accomplished by first dissecting and separating placental tissue. Next, the cells of the olein are homogenized.
Then the organelles are separated by several centrifugation steps. Finally, the floating layer containing the lipid droplets is collected and the lipid drops are characterized. Ultimately, fluorescence microscopy and western blood analysis are used to show the purity of the lipid droplet fraction.
The main advantage of this technique is that it is possible to isolate lipid droplets from most aortic cells. Note that in certain tissues, the number of droplets can below those reducing the floating G layer and the number of droplets. Placentas were collected from healthy women with singleton pregnancies undergoing elective cesarean section delivery prior to the onset of spontaneous labor at term subjects gave written informed consent for the collection of their placenta.
The collection and subsequent use of placentas was performed with approval from the University of Tennessee and University of Tennessee Graduate School of Medicine in Knoxville. Institutional review board in the biological safety hood, transfer the placenta to a sterile autoclavable container and carefully wash blood from placenta and membranes using sterile saline. Discard the bloody saline in a one liter beaker as liquid biological waste.
Place the placenta on a sterile field with this smooth surface bearing the umbilical cord facing up with sharp, fine point scissors and forceps. Remove the umbilical cord and fetal membranes. Next, flip the placenta over so that the maternal surface is facing up.
Then remove the overlying basal plate tissue about three millimeters from the surface. Dissect one COTA leadin at a time, avoiding the chorionic plate, and collect the villous tissue into a 250 milliliter beaker with saline. Then use 0.9%sterile saline and forceps to rinse the tissue several times in a separate beaker by swirling using the one liter beaker for liquid waste.
Transfer one codi leadin at a time to 150 millimeter Petri dish. Hold with forceps and use a razor blade to scrape the tissue from the vessels onto a Petri dish. Place the scraped tissue into a separate beaker containing 0.9%sterile saline.
Repeat for all tissue pieces. After rinsing the scraped tissue in cell dissociation cve, drain it of excess liquid and weight to digest the placental tissue. After preparing the tissue digestion mixture, divide the tissue transferring 60 grams from total collected tissue to a 500 milliliter sterile Helen Meyer flask.
Add the tissue digestion mixture to the flask containing the tissue and incubate at 37 degrees Celsius in a shaker for 45 minutes. At 150 RPM to collect the dissociated cells after the incubation and tilting the flask to allow the tissue to settle. Collect the super natin.
Taking care not to collect UND disassociated tissue. Add an equal amount of HBSS to the supernatant before transferring into sterile 15 milliliter centrifuge tubes. Repeat the dissociation and supernatant collection with the remaining portions of UND associated tissue for each batch following centrifugation at 1000 times.
Gravity for 15 minutes at four degrees Celsius aspirate the supernatant without disturbing the pellet. The placental villus cells are predominantly in the white portion of the pellet overlying the red blood cells. Transfer the villus cells in the white portion of the pellet to a sterile 50 milliliter conical centrifuge tube and keep on ice.
After collecting the cells from all three digestion stages, use a 100 micron nylon cell strainer inserted in the top of a sterile 50 milliliter conical centrifuge tube to filter the suspension. If the filtration of the cell suspension slows lift upward on the filter to draw a vacuum within the tube centrifuge at four degrees Celsius and 1000 times gravity for 10 minutes. Carefully remove the supernatant without disturbing the pellet to homogenize placental villus cells using freshly prepared hypotonic lysis medium or HLM in a biological safety hood, add four times the cell pellet volume of ice cold HLM to the cells.
Use a 10 milliliter pipette to gently and thoroughly resus. Suspend the cells by pipetting them up and down. After incubating the cells on ice for 10 minutes, transfer them to an ice cold down homogenizer and slowly homogenize the cells with the loose fitting pestle by applying 20 to 25 gentle strokes.
Spin the lysate at 3000 times gravity and four degrees Celsius for 10 minutes to remove unbroken cells. Then collect the supernatant into a new tube before spinning again at 25, 000 times gravity and four degrees Celsius for 20 minutes. To remove the mitochondria to isolate the lipid droplets by ultracentrifugation, collect the super natin in a 50 milliliter centrifuge tube.
Adjust to 20%sucrose and transfer into the bottom of an ultracentrifuge tube for an S SW 28 rotor or equivalent overlay the density adjusted super natin with about 10 milliliters of ice cold, 100 millimolar sodium carbonate buffer and 0.5 to one milliliter of ice cold HLM to fill the tube using an S SW 28 swinging bucket rotor centrifuge at 130, 000 times gravity and four degrees Celsius for 45 minutes. Then collect the floating layer, adjust to 10%sucrose and transfer it to the bottom of a 13.2 milliliter ultracentrifuge tube for an SW 41 TI rotor or equivalent overlay the density adjusted supernatant with about five milliliters of ice cold, 100 millimolar sodium carbonate buffer and 0.5 milliliters of ice cold HLM following centrifugation at 274, 000 times gravity and four degrees Celsius for 60 minutes. In an SW 41 TI rotor, use a one milliliter pipette to carefully collect the top floating layer containing lipid droplets.
Adjust the volume to 10%sucrose and again transfer to a 13.2 milliliter ultracentrifuge tube before overlaying with five milliliters of ice cold, 100 millimolar sodium carbonate buffer and 0.5 milliliters of ice cold HLM. After centrifugation for 30 minutes, use a bent PE pipette to carefully collect the top white colored floating layer containing the lipid droplets into three 1.5 milliliter tubes containing 100 microliters of HLM. Characterize the lipid droplets according to the text protocol as seen in this figure, the presence of lipid droplets isolated from human term placental villus cells was verified by staining with a neutral lipid specific fluorescence dye boda P 4 93 5 0 3 and visualized using fluorescence microscopy.
Shown here, the purity of the isolated lipid droplet fractions was evaluated by western blotting with marker proteins for lipid droplets that included para lippin two calnexin for the er, the goi marker, GM one 30 CO X four for mitochondria and plasma membrane, MEK one. After dilapidating, the lipid droplets with cold acetone and the proteins extracted. The fractions were subjected to Western blotting para lipin.
Two, the lipid droplet protein was detected in post nuclear supernatant and in the isolated white floating layer containing lipid droplets. Proteins specific for plasma membranes such as MK one and GM one 30 were not detected in lipid droplet fractions in either layer beneath the floating layer for spin four and spin five. As previously reported, the protein calnexin and a weak staining of mitochondrial membrane protein CO X four have been detected elsewhere in the lipid droplet fraction.
These results are consistent with earlier reports showing that lipid droplets interact with mitochondria in mammalian cells and with the ER r. Following this procedure, other methods like proteomic and lipidomic methods can be performed to study additional questions like what factors are bound to the droplets.
