The overall goal of this procedure is to prepare dendritic cells for imaging studies using Q dot nano crystals. This is accomplished by first isolating bone marrow cells from murn, femurs, and tibias. Next bone marrow cell cultures are set up to facilitate cell differentiation into dendritic cells in the presence of GM CSF.
Then the dendritic cells are recovered from the bone marrow cell cultures after eight days. The final step of the procedure is to stain the cells with fluorescent Q nano crystals. Ultimately, brightly stained dendritic cells can be visualized by fluorescence microscopy.
The main advantage of using this technology compared to other methods like using GFP tag dendritic cells, is that Q dot nano crystals retain their fluorescence even after staining with organic solvents like acetone. This method can help answer key questions in dendritic cell biologists, such as a migration properties of these cells in different diseases or in the context of different diseases. Demonstrating the staining procedure will be Maria Ali, a graduate student from my lab.
After sacrifice, carefully dissect the tibia and femurs from two mice without cutting the bone ends. Then use tissue paper to clean all the attached tissues from the bones, being careful not to break them. In a biosafety hood.
Fill a 35 millimeter Petri dish with 70%ethanol, and then immerse the bones in the ethanol for 10 minutes. After the bones have been sterilized, recover them from the ethanol and let them air dry for five minutes in a new Petri dish. Now, cut the femur in the middle and the tibia by their thinnest tip.
Infuse the insides of the bones with one milliliter of RPMI medium using a sterile syringe. Collect the cell suspension in a 15 milliliter tube and wash it two times with RPMI medium by a 10 minute centrifugation at 300 times G at four degrees Celsius in a centrifuge with a swinging bucket rotor. After the last wash, resus suspend the cells in two milliliters of a CK lysis, buffer, and incubate for five minutes at room temperature in order to eliminate red blood cells.
Next, add 13 milliliters of RPMI plus 10%FBS and wash the cells two more times in this medium with the same centrifuge settings as before. Count the cells and adjust the concentration to two times 10 to the fifth cells per milliliter. With RPMI plus 10%FBS add recombinant murine G-M-C-S-F to a final concentration of 20 nanograms per milliliter.
Then add 10 milliliters of this suspension to a sterile microbiological quality 10 centimeter Petri dish and culture. The cell suspension in a carbon dioxide incubator. Three days later, add another 10 milliliters of RPMI plus 10%FBS with 20 nanograms per milliliter of recombinant murine G-M-C-S-F to each of the prepared plates.
Three more days later, recover 10 milliliters of cell suspension from each Petri dish, centrifuge the cells under the same conditions and resuspend the pellet in the same volume of RPMI plus 10%FBS as recovered from the Petri dishes and replenish with new recombinant murine G-M-C-S-F. Finally, return the cell suspension to each original Petri dish and culture the cells for two additional days in the carbon dioxide incubator. After eight days in culture, recover the floating and loosely adherent cells by washing the Petri dishes with fresh medium.
Then label the collected cells with the Q tracker 6 55 cell labeling kit strictly following the manufacturer's instructions to label five times 10 to the sixth of the murine bone marrow derived dcs with Q nano crystals at a 10 nano molar concentration. First mix five microliters of each of the kit components A and B in an einor tube, and incubate the mixture for five minutes at room temperature. Then add one milliliter of RPMI plus 10%FBS, and vortex the tube for 30 seconds.
Next, add five times 10 to the fifth. Dcs suspended in 0.5 milliliters of RPMI plus 10%FBS to the einor tube and incubate the cells in the D solution at 37 degrees Celsius for 60 minutes. Then wash the cells two times in RPMI plus 10%FBS under the same conditions as before, and resuspend the pellet in RPMI.
This figure shows an example of a fluorescent micro photograph of dcs. Immediately after labeling, a drop of labeled cells was deposited on a glass slide and covered by a cover slip. Then the samples were evaluated using a fluorescent microscope.
In this figure, a fluorescent micro photograph of dcs after 48 hours, maturation in vitro can be seen labeled. Dcs were cultured for 48 hours with 100 nanograms per milliliter, LPS and 20 nanograms per milliliter, TNF alpha on slide culture chambers in a carbon dioxide incubator. Then the samples were washed with PBS fixed with acetone, counterstain with DPI and evaluated using a fluorescent microscope.
Labeled dcs matured in vitro were analyzed by flow cytometry Q dot stained cells give a strong signal in the FL three Channel Q do stained and unstained. Dcs were additionally stained with specific antibodies against maturation markers, CD 86 and CD 40, and isotype controls cells were then analyzed by flow cytometry. Additionally, IL six and nitric oxide levels were determined in supernatants of mature Q stain DCS and controls.
Here a representative figure of labeled DCS in a dissected tissue is shown labeled. Mature dcs were intravenously injected into C 57 black six mice. Three days later, the mice were sacrificed and the spleens were collected.
Snap frozen embedded in OCT and eight micron sections were prepared using a cryostat. The samples were then fixed with acetone, counterstain with DPI, and evaluated using a fluorescent microscope. Following this procedures.
Other methods like antigen posing or maturation with different stimuli can be performed in order to answer additional questions such as the migration of dendritic cells in response to different maturation stimuli.
