Isolatie en intraveneuze injectie van muizen beenmerg afgeleide Monocyten

Hello, my name is Helen Kuster and I'm working at the cardiovascular research Group of Dr.Harold and professor at the Auto Vica University in MacBook. We are interested in the isolation characterization of marine monocytes in order to use for vehicles in collateral vessel growth. The following major steps are necessary for the cultivation of bone marrow derived monocytes, first mouse anesthesia and cervical dislocation, second extraction and rinsing of the femur.

Third filtering of the bone marrow. Fourth to washing steps with medium fifth, cultivation of the cells on ultralow attachment plates. Sixth flow cytometry.

In order to extract monocytes from the bone marrow, the mouse will be anesthetized by using ISO fluorine and euthanized by cervical dislocation. The mouse will be fixed and both lower limbs are separated with a steroid scalpel. Both femur and tibia were harvested and washed with 96%Ethan ethanol for a minimum of 90 seconds.

After this, all steps must be strictly sterile to avoid contamination. Muscles or sinew will be removed from the bone. Femur and tibia are rinsed with warm PBS for several times.

For harvesting the bone marrow, the proximal and distal ends of each bone are cut with a pair of fine scissors. In order to rinse the bone, we need one steroid 28 G needle, one, one milliliter syringe, and between 10 to 15 milliliter of medium per bone. Afterwards the bone marrow is filter through a 70 micrometer S while holding the bone with a fine forceps.

Rinse the bones one by one until they turn illusioned. In order to minimize cell stress, carefully apply pressure to the stamp of the syringe. Rinse the siet with PBS.

Afterwards, the cells are centrifuged at 250 G for 10 minutes at room temperature. And are reus suspended with medium? After this step, repeat the step once again.

For bone marrow derived monocytes from native bone marrow, we use M 199 with 1%penicillin and streptomycin and 10%fetal cough serum. Additionally, we add 20 nanograms per milliliter INE MCSF on the first day. Eight nanogram per milliliter interfer gamma are added on the last day of culture to the medium.

If MHC two upregulation is wished, the cells are seeded into ultra-low attachment plates with thick wells to prevent permanent adhesion onto the bottom. We use a concentration of one time 10 to the six cells per milliliter with up to six milliliters per well. The cultivation period is five days at a temperature of 37 degrees and 5%carbon dioxide.

The cells are checked daily after five days between 60 up to 80%of the cells are monocytes. My name is Martin Bachner and I'm a medical doctor student here at Auto Fungi University in at the Department of Cardiology, anology, and chronology. And when you do the intravenous injection, it is important to practice a lot before doing it because otherwise you won't be able to apply the monocytes in an adequate way.

Experiments have shown that monocytes are most effective in improving art agenesis when applied systemically. A common way of systemic drug application is a tail WA injection. In our case, we inject 2.5 million monocytes, which are resuspended and sodium chloride.

There are four vessels in the tail of the mouse arteries at the ventral and dorsal side, and two wanes on the lateral side. So for WA injection, we have to turn the tail for about 90 degrees. For easier procedure, the mouse should be warmed by using a heating pad for about 10 minutes.

The animals should be observed by warming. This is important for recognizing signs of overheating. Furthermore, we need this infection agent and a 30 gene needle set on a one milliliter insulin syringe.

Fix the animal carefully in a restrainer, which makes it much easier to inject into the tail way. Make sure the mouse gets enough space for breathing before loading the solution into the syringe. Vortex, the monocyte solution to make sure that all monocytes can be injected.

Disinfects injection site to avoid infection, take the tail between your thump and your index finger. At the most distal point, the needle is inserted in a flat angle. If it is placed in the vein, it is very easy to inject the monocyte solution.

If there's a blister, it is a sign of a wrong injection. You should stop immediately and try more. Proximal, always try to inject slow and not more than five microliters per gram.

If the injection was successful, stop the bleeding at the injection site by applying gentle pressure for about one minute. At the end of the procedure, open the retractor and place the mouse in its cage to characterize monocytes. We use the effects with a combination of antigens, namely CD 11 B, F four 80, CD 115, and GL one.

The expression of these antigens correlates with a stage of development for analyzing the monocytes. We did the facts on day 0, 3, 5 and seven, the monocyte purity increases up to 90%on day five. To achieve that purity, it is important to deplete strongly terrine macrophage.

Non terrine cells are depleted by using EDTA free wash buffer. To deplete terrine cells, we're using EDTA containing wash, buffer and gentle p petting. This diagram shows the main cell types for their one end culture.

The different cell types shown are one lymphocytes, two monocytes, three granulocytes, increasing monocyte purity after day five. And culture. Note, the shifting cellular composition during cultivation cell population includes one monocytes and two macrophages.

Figure two, timeline of CD 115 expression during differentiation. Note that over 95%of all cells are CD 115 positive after five days of differentiation, indicating that the vast majority of cells are either monocytes or macrophage. Classification can be based on cell size and granularity, but specific cellular markers are more reliable.

Figure three, increasing expression of monocyte macrophage maturity. Marker F four 80, day five, note the appearance of a population with intermediate F four 80 expression, which is consistent with monocyte phenotype. Day seven, note the right shift of F four 80 expression consistent with macrophage maturation.

The isolation of monocytes is important in essential for many in vitro and in vivo studies. These cells are an interesting target, for example, in connection with diseases like peripheral arterial disease or coronary heart disease, which are related to ischemia. Bone marrow from FEMA and tibia of biopsy mice is harvested by flushing bones with warm medium cell suspension is supplemented with MCSF and cultured on ultra low attachment surfaces to avoid adhesion triggered differentiation of monocytes properties and differentiation of monocytes is checked at different times and facts with marker like CD 11 b, CD 115 and FO 80 is used to phenotype the cells.

If systemic drug delivery is desired, the intravenous injection is an easy and effective way to do so. We described the method to isolate large amounts of MI monocytes from bone marrow in a simple and cost efficient way. With this new method, we're able to generate about 11 million monocytes per mouse.