Osteoclast Afleiding van Mouse Bone Marrow

The overall goal of this procedure is to isolate osteoclasts from mouse bone marrow in large numbers. This is accomplished by first harvesting bones from euthanized mice. Next, the cells are liberated from the bone marrow by using a mortar and pestle to crush the bones in facts buffer.

Then the bone marrow solution is layered onto the density gradient cell separation medium and density gradient cell separation is carried out. Finally, the layer containing the cells of interest is aspirated and the cells are placed in bone marrow microphage induction medium. Ultimately trap staining is used to show the presence of a large number of osteoclasts.

The implications of this technique extend toward diseases associated with altered osteoclast function, which can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis to more commonly encounter pathologies such as osteoporosis because it produces a reliable method of isolating a large number of osteoclasts from mouse bone marrow. To begin, bring 10 milliliters of commercially available density gradient cell separation, medium to room temperature in a 50 milliliter conical tube. Place a 50 milliliter aliquot of fax buffer at room temperature to be used with the density gradient cell separation medium.

After preparing additional media and euthanizing a C 57 black six mouse, according to the text protocol, position the mouse on a dissection board and use 70%ethanol. To spray it, harvest the fera tibia hum eye and spine. Then place the bones in facts buffer on ice.

Next, using clean tissue paper, remove the muscle and soft tissue from the bones. Place the bones into a mortar and pestle with three milliliters of facts buffer and gently crush them. Then aspirate the bloodstained fluid and pass it through a 70 micrometer strainer into a 50 milliliter conical tube.

Add an additional five milliliters of effects buffer to the crushed bone and crush further before transferring the liquid through the strainer into the 50 milliliter tube. Continue with this process until the fluid no longer stains red before pelleting the bone marrow at 200 Gs for five minutes at four degrees Celsius. To perform a gradient separation, aspirate the supernatants from the cell pellet and use 10 milliliters of fax buffer to resus.

Suspend the pellet, tilt the conical tube of room temperature density gradient cell separation medium to about 30 degrees, and using an electric pipette with the tip of the pipette against the inner top surface of the tube. Slowly layer the bone marrow solution onto the medium using a room temperature centrifuge with no acceleration or deceleration. Spin the gradient at 200 Gs for 15 minutes.

Then aspirate the cloudy middle layer, which contains the cells of interest and transfer them into a new conical tube. Add 20 milliliters of ice cold fax buffer to wash the cells. Then use one milliliter of bone marrow microphage stimulating medium to resuspend the final cell pellet following cell counting.

Placed two milliliters of microphage stimulating medium into each well of a 24 well plate. Then add 200, 000 cells to each well, a cell density that will ensure adequate in vitro osteoclast culturing. Gently agitate the plate and incubated at 37 degrees Celsius without changing the medium for three days.

After the third day, change the medium to bone marrow osteoclast induction medium and begin to change it daily for five to seven days. At which point large multinucleated osteoclasts should be visible. Refer to the text protocol for staining and the resorption assay as seen here.

A high number of osteoclasts can be confirmed using tartrate resistant assay. Phosphotates staining osteoclasts are visualized as large purple cells with multiple nuclei using the protocol demonstrated in this video. It is common to isolate osteoclasts with as many as 30 nuclei per cell.

Using a mineralized surface is considered the gold standard method for assessing osteoclast resorptive activity in vitro. In this figure, brightfield microscopy at 10 x magnification demonstrates a percentage of mineralized surface or resorptive pits that are formed as outlined in black, which allows for determination of osteoclast resorptive capacity. After watching this video, you should have a good understanding of how to reliably isolate a large number of osteoclasts from mouse bone marrow.