The overall goal of this procedure is to isolate and culture pulmonary endothelial cells from neonatal mice. This is accomplished by first creating a homogeneous cell suspension from freshly harvested lungs. Finally mince the tissue and subjected to enzymatic digestion, then enriched for endothelial cells using anti P chem one antibody conjugated magnetic beads.
CETA culture purify the endothelial cell population. Further by using anti I chem two antibody conjugated magnetic beads and amplify these cells in a gelatin coated flask. The resulting primary pulmonary endothelial cells are mostly of microvascular origin and express specific endothelial cell markers such as TERRIN and VEGF R two.
The main advantage of this technique over existing methods such as irna gene silencing, is that it allows utilization of transgenic mice to obtain genetically modified endothelial cells. Furthermore, endothelial cells obtain using this technique are mostly of microvascular origin, and as such may be more relevant to some areas of vascular research. Endothelial cells obtained with this method can help answer key questions in the field of vascular biology, such as those regarding molecular mechanisms of angiogenesis and vascular permeability.
Visual demonstration of this technique is critical as the tissue harvest digestion and bead bound cell washing steps are difficult to learn. Improper performance of this technique can lead to non endothelial cell contamination and poor cell yield and growth. For this dyna beads protocol prepare 50 milliliters of 0.1%BSA in PBS and mix by vortexing until the BSA dissolves then sterilize the solution by passing it through a 0.22 micron syringe.
Filter Under sterile conditions in the tissue culture hood Resus suspend sheep, anti rat IgG dyna beads and transfer 200 microliters into a 1.5 milliliter einor tube. In order to wash the beads, mount the tube on the magnetic particle concentrator or MPC and allow one minute for the beads to sediment aspirate the supernatant. Remove the tube from the MPC and resuspend the beads in one milliliter of sterile 0.1%B-S-A-P-B-S.
Repeat this wash three more times after the fourth wash. Remove the tube from the MPC and Resus. Suspend the beads in 500 microliters of 0.1%B-S-A-P-B-S.
Add 10 microliters of rat anti mouse pcam one antibody to the tube and tumble for two hours at room temperature as shown earlier. Wash the beads four times with sterile 0.1%B-S-A-P-B-S. Remove the tube from the MPC and resuspend.
The beads in 200 microliters of 0.1%B-S-A-P-B-S store. This preparation of anti P chem one antibody conjugated dyna beads at four degrees Celsius. To obtain primary cells, anesthetize three, six to eight day old pups.
Ensure effectiveness of the anesthesia by pinching the tails with forceps. Dissect animals sequentially. First pin the pup to aboard and remove the skin from the chest area.
With dissection scissors excise the rib cage to expose the lungs. Now aseptically excise individual lung lobes. Being careful not to dissect the bronchi or any visible connective tissue around the lungs.
Combine all lungs in ice cold DMEM finally euthanize the pups. Now move to the tissue culture hood. Remove the lung lobes from DMEM and place them in a 100 millimeter tissue culture dish.
Aspirate excess DMEM with sterile scissors mince the tissue. Finally by cutting about a hundred times for enzymatic digestion. Transfer the tissue preparation into 15 milliliters of warm collagenase disc space solution and incubate for 45 minutes at 37 degrees Celsius on a rotator with a 20 milliliter syringe connected to a 14 gauge cannula T rate clumps of the digested tissue into a single cell suspension.
Now pass the cell suspension through a 70 micrometer cell strainer and wash the strainer with 15 milliliters of Im to stop the digestion reaction. To harvest the pulmonary cells centrifuge at 400 Gs for five minutes and aspirate off the supernatant. Then resuspend the cell pellet in three milliliters of 0.1%B-S-A-P-B-S and transfer to a five milliliter round bottom polystyrene tube.
To isolate the endothelial cells at 22.5 microliters of anti P chem one antibody conjugated dyna beads and tumble at room temperature for 12 minutes. In the meantime, coat a T 75 flask with two milliliters of 2%gelatin. Aspirate the excess gelatin solution and allow it to air dry inside the tissue culture hood for about half an Once the bead incubation is complete, split the cell suspension equally into three fendor tubes and mount on the MPC.
Using the MPC system, wash the dyna beads five times with about one milliliter of 0.1%B-S-A-P-B-S After the final wash, re suspend the beads in one milliliter of VAs life per tube. Mix well combining all tubes. Plate the dyna bead cell preparation on the pre-coded T 75 flask and bring the total volume in the flask to 10 milliliters with VAs life.
Now place the flasks in a 37 degree Celsius 5%carbon dioxide incubator. Change media the following day, allow one full day of growth and then change half of the media every other day until the cells are greater than 70 to 80%confluent. And to further purify the primary cell culture, prepare antibody conjugated dyna beads as shown earlier, but this time using CD 1 0 2 and muse I chem two antibody.
Also coat a T 75 tissue culture flask with 2%gelatin as shown earlier. Now remove the T 75 flask with cells from the incubator. Aspirate the media and wash the primary cells with eight milliliters of PBS aspirate.
The PBS add two milliliters of 0.05%tripsin EDTA and allow about five minutes for cells to detach. Tap the flask lightly to eight cell detachment. Add two milliliters of IM to inhibit the trypsin and scrape any remaining adherent cells.
Transfer the cell suspension to a 15 milliliter tube. Harvest cells by centrifugation for five minutes at 400 GS aspirate off the media and resuspend the cell pellet in two milliliters of 0.1%B-S-A-P-B-S transfer to a five milliliter polystyrene round bottom tube and add 10 microliters of anti icam. Two dyna beads tumble for 12 minutes At room temperature, split the cell suspension into two 1.5 milliliter einor tubes and mount on the MPC system.
As before, wash the beads with one milliliter of 0.1%B-S-A-P-B-S-A total of five times. Now resus suspend each tube in one milliliter of VAs life and perform a cell count. Finally, plate the cells at 800 to 1000 K cells per T 75 flask in 10 milliliters of VAs life Place the flasks in a cell culture incubator.
Change the media every other day until the cultures reach about 80%co fluency. The purified mouse lung endothelial cells are ready to be used in experiments. Typically, a cell preparation from three six to eight day old pups yields 1.2 to 1.5 times 10 to the sixth pulmonary endothelial cells after a weak in culture.
As expected, these primary endothelial cells display cobblestone morphology under light microscopy and also show VE coherent staining at cell to cell junctions. Using this protocol, the majority of mouse lung endothelial cells express VE coherent and VEGF R two as demonstrated by fax analysis. The red line indicates isotype specific control and the green line indicates anti VE or anti-VEGF R two specific IgG.
When attempting this procedure, it's important to harvest only the target tissue, avoid over digesting the cells and wash the bead bound cells thoroughly. Endothelial cells obtained. Using this procedure can be used for functional assays in the field of vascular biology to analyze responses relevant for angiogenesis and vascular permeability.
After watching this video, you should have a good understanding how to isolate primary mouse endothelial cells using chem one antibody and chem two antibody bound dyna beeps, and how to obtain pure endothelial cell cultures.
