The overall goal of the following procedure is to examine the ability of purified hematopoietic progenitors to give rise to plasmacytoid dendritic cells in intestinal pire patches upon adoptive transfer. This is accomplished by first performing hydrodynamic gene transfer of the plasmid, encoding the dendritic cell growth factor. FLIT three ligand into PI's patch plasmacytoid dendritic cell impaired mice.
In the second step, common dendritic progenitor cells are isolated from wild type bone marrow by magnetic bead separation and flow sorting. In the final step, the progenitor cells are adoptively transferred by intravenous injection into pyres patch plasmacytoid dendritic cell impaired mice that were treated by hydrodynamic gene transfer. Ultimately, the donor hematopoietic progenitor cell derived PI's patch plasmacytoid dendritic cell population can be evaluated by flow cytometry.
This method can help answer key questions in the immunology field, such as what is the developmental origin of the plasma Cy Dendri cells that localize within the pirate patches. Though this method can provide insight into the origin of pyres patch plasmacytoid dendritic cells. It can also be applied to understanding how plasmacytoid dendritic cells affect the lymphocyte populations within these tissues.
Two days before adoptive transfer dilate the blood vessels of five CD 45 2 positive interferon alpha receptor knockout recipient mice under a heat lamp for five to 10 minutes. Then for each mouse, place the animal in a restrainer and disinfect the tail with 70%ethanol. Next, use a 27 gauge needle to inject five micrograms of plasma and coating flit three ligands and two milliliters of sterile PBS into the tail vein of each animal.
For the control cohort, inject five micrograms of empty vector. After monitoring the mice for 15 to 30 minutes, return them to their housing facility. On the day of the experiment, place the dissected femur and tibia from 10 congenic CD 45 1 positive C 57 black six mice into a culture dish containing complete media.
Then insert a 27 gauge needle into one end of each bone and use complete media to flush the bone marrow into the dish three times per bone. Ensure that the bone marrow cells are being removed by confirming that the expelled media appears cloudy. Make a single cell suspension by gently pipetting the media up and down three to five times in the culture dish.
Then remove the debris by passing the bone marrow cells through a 40 micrometer cell strainer and disrupt any cell clumps by gently applying the end of a sterile syringe plunger. On the strainer, spin down the cells, remove supernatants and lice the red blood cells in two milliliters of lysis buffer per four to six times 10 to the seventh cells for five minutes at room temperature. Then add 10 milliliters of fax buffer to the bone marrow cells and spin down for four minutes at 500 times G at room temperature.
After gently aspirating the sup natin, wash the pellet in 10 milliliters of fax buffer. Next, stain the cells with hematopoietic lineage marker recognizing antibodies following antibody staining and washing. Incubate the cells with goat anti rat magnetic microbeads.
During the microbead incubation, load a max LD column onto a MIDI max cell separator and pre rinse the column with two milliliters of fax buffer. Then place a 15 milliliter conical tube under the column and load up to five times 10 to the eighth cells in 2.5 milliliters of fax buffer to the column. Wash the column three times with two milliliters of fax buffer per wash, collecting the lineage negative cells that pass through the column into the 15 milliliter tube.
Count the eluded cells. Then after staining the cells with the appropriate fluorescently conjugated antibodies, transfer the sample to a fax tube for sorting the common dendritic progenitors on the basis of the desired marker profile. Collect the fax purified cells in a 15 milliliter tube containing five milliliters of complete media, and then at the end of the fax run record the absolute number of sorted cells.
After spinning down, the cells dilute one times 10 to the fifth of the purified common dendritic progenitors per 100 microliters total volume of sterile PBS per mouse and aspirate the cell suspensions into one syringes equipped with 27 gauge needles. Then inject the CD 45 1 positive progenitor cells into the tail vein of each recipient CD 45 2 positive interferon alpha receptor knockout mouse as just demonstrated seven to 10 days following the adoptive transfer. Remove the intestines from the common dendritic cell progenitor injected mice placing the tissues onto PBS soaked paper towels.
Then use a pair of fine forceps and scissors to collect all of the visible pyres patches along the wall of the small intestine. Note that the pyres patches are often structurally distinct even within a single mouse, so take care to ensure that all of the P'S patches are identified and dissected. Wash the isolated P'S patches three times in a Petri dish containing PBS using forceps to remove the feces.
Then digest the P'S patches with one milligram per milliliter, collagenase four in 10 milliliters of HBSS in a 50 milliliter flask with vigorous stirring at 37 degrees Celsius. After an hour, pour the digested PIs patches into a 40 micrometer cell strainer and use a sterile syringe plunger to force the cells into a 15 milliliter conical tube. After spinning down the filtered cell suspension, resuspend the pellet in six milliliters of 37%per call solution and transfer it to a 15 milliliter tube.
Then gently place six milliliters of 70%per call solution underneath to form a per call step gradient. Now separate the cells for 20 minutes at 800 times G at room temperature with the break off. Then collect the mononuclear cell population at the interface.
After pelleting the collected cells, wash them twice in 50 milliliters of complete media. Then stain them with the appropriate antibodies to detect the CD 45 1 positive PYS patch. Plasmacytoid dendritic cells that derive from the adoptively transferred CD 41 positive progenitors.
Analyze the cells by flow cytometry. The plasmacytoid dendritic cells can be identified by their CD 11 C positive clec H positive PDCA one positive B two 20 positive CD 11 B negative phenotype. Finally determine the absolute number of plasmacytoid dendritic cells using this equation.
These dot plots indicate the gating strategy for the isolation of common dendritic progenitors. For mouse bone marrow cells as just demonstrated common dendritic progenitors comprise approximately 0.1%of the total bone marrow cells and roughly four to six times 10 to the fourth. Of these cells can be isolated from one mouse to identify PI's patch plasmacytoid dendritic cells by flow cytometry.
An initial forward and side scatter gating strategy is used followed by gating for CD 11 C positive clec H positive cells. Interferon alpha receptor knockout mice exhibit a significant reduction in pys patch plasmacytoid dendritic cells relative to wild type mice. Common dendritic progenitor cells also differentiate into conventional dendritic cells.
In contrast to plasmacytoid dendritic cells CD 11 C positive clec H negative conventional dendritic cells are observed in similar amounts in both wild type and knockout mice. Hence interferon alpha receptor knockout mice provide an opportunity to examine PS patch plasmacytoid dendritic cell reconstitution without the effects of lethal irradiation. The adoptive transfer of wild type common dendritic progenitors into interferon alpha receptor knockout mice as just demonstrated stimulates an increase in PIs patch plasmacytoid dendritic cells.
Moreover, pretreatment with FL three ligand hydrodynamic gene transfer further enhances plasmacytoid dendritic cell expansion in PIs patches implying that transferred common dendritic progenitors and possibly endogenous FL three positive progenitors respond to FL three ligand by inducing pyrus patch plasmacytoid dendritic cells. Both conditions also stimulate CD 11 C positive clec H negative conventional dendritic cell production consistent with the developmental origin of conventional dendritic cells. PYS patch plasmacytoid dendritic cells expressed traditional plasmacytoid dendritic cell markers including clec H, PDCA one and B two 20 and LAC CD 11 B.In addition, analysis of the PYS patches from interferon alpha receptor knockout mice that receive both FL three ligand hydrodynamic gene transfer and transferred common dendritic progenitors shows that about 70%of the plasmacytoid dendritic cells are derived from donor CD 45 1 positive common dendritic progenitors.
Collectively, these data demonstrate that adoptive transfer of common dendritic progenitors induces the PIs patch plasmacytoid dendritic cell population in response to FL three ligand mediated signals in vivo. After watching this video, you should have a good understanding of how to isolate and identify pars patch plasmacytoid D cells.
