This video demonstrates a protocol for isolation and phenotypic characterization of hematopoetic progenitors using magnetic bead based separation and fluorescence activated cell sourcing. First, the hind leg, femur, tibia, and spinal cord are dissected from a mouse and homogenized to generate a bone marrow cell suspension. The population is then enriched for cells that express C kit using magnetic microbeads flow.
Cytometric analysis of the enriched population demonstrates that this method yields highly purified hematopoietic lineage, progenitors and hematopoietic stem cells, which can then be used for in vitro and in vivo functional studies. The main advantage of this technique over existing methods like bone flushing, is that it allows more efficient and faster recovery of bone marrow cells. The implications of this technique extend toward the therapy or diagnosis of inflammation, autoimmunity, immunodeficiencies, degenerative disease, metabolic disorders, and cancer immune models, because it allows the phenotypic characterization and ex vivo functional analysis of hematopoietic progenitors.
So this method can provide insight into monitoring hematopoesis in different physiological and pathological condition. It can also be used to study the cell biology signal transaction and developmentally regulated gene expression in distinct progenitor subsets. Begin by placing a euthanized mouse in a stainless steel pan.
Spray the abdomen and back with 70%ethanol to collect the bones that will be used as a source of cells. Use scissors to make two small openings in the skin at the hind legs with the help of forceps dislocate the femur, then turn the mouse upside down and cut the skin longitudinally. To display the spinal column, remove the spinal column.
Then using sharp tip scissors. Carefully remove all soft tissue residues from the spinal column. Use gauze to remove soft tissue residue from the femur and tibia.
Then place the bones in a 50 milliliter conical tube containing 30 milliliters of supplemented RPMI.Medium. Place several four-fifths sterile filters in a sterile mortar. Then using forceps, transfer the bones into the mortar and cover them with another layer of filters to assemble a friction surface.
Next place a 40 micron cell strainer filter in a 50 milliliter conical tube. Then using a pestle, smash the bones until a homogenous cell suspension is obtained. And using a pipette, transfer the suspension into the cell filter.
Next, add five milliliters of fresh supplemented RPMI to the mortar to wash and harvest the remaining cells pipette up and down. Then add the RPMI to the tube with the filter. Repeat the last two points until the homogenized bones appear clear.
Once the solution has passed through the filter, transfer it to a new 50 milliliter tube and filter and repeat the process to eliminate the remaining tissue debris.Centrif. Use the tube at 500 times G for five minutes at four degrees Celsius and carefully remove the supinate by aspiration. Make sure not to disturb the cell pellet.
The next step of the procedure is to characterize the phenotypes of hematopoietic progenitors. Begin by Resus suspending the cell palate from each mouse in 15 milliliters of PBS and 2%FBS. Then centrifuge the tube at 500 times G for five minutes at four degrees Celsius and carefully remove the supinate by aspiration.
Do not disturb the pellet. Reese has bend the pellet in five milliliters of PBS and 2%FBS and count the cells using a hemo cytometer. Once the cell concentration has been determined, transfer one time cent of the six cells to each well over a 96 well round bottom plate.
Then centrifuge the plate at 500 times G for five minutes. During the spin, prepare a mix of monoclonal antibodies directed against the surface markers listed here in PBS and 2%FBS to stain the cells. Once the spin has completed, discard the supinate by inverting the plate and add 50 microliters of the antibody mix to each well.
Then using a multichannel pipetter pipette up and down to dissociate into single cell suspensions. Incubate the cells for 20 minutes in the dark at four degrees Celsius following the incubation wash cells twice with 150 microliters of PBS and 2%FBS. Then centrifuge the plate at 500 times G for five minutes.
Then discard the senna by inverting the plate. Next, add 50 microliters of diluted one to 300 AP three size seven strept adin in PBS with 2%FBS to each well dissociate into single cell suspension by pipetting. Incubate the cells for 10 minutes in the dark at four degrees Celsius following the incubation.
Wash the cells twice with 150 microliters of PBS and 2%FBS After centrifuging the plate to gain discard the S supernatant and re suspend the cells in 100 microliters of PBS and 2%FBS analyze the samples by cytometry. Next to sort selected subsets of hematopoetic cells, magnetic enrichment is performed on bone marrow to enrich secret positive cells. Reeses spend the cell pal of bone marrows derived from at least 10 mice in one milliliter of buffer solution containing C kit monoclonal antibody conjugated to a PC diluted one to 50.
Incubate the cells for 20 minutes in the dark at four degrees Celsius following the incubation. Wash the cells twice with 40 milliliters of buffer solution. After centrifuging, carefully remove the supine natum by aspiration.
Then add 50 microliters of anti A PC microbeads per mouse directly to the pellet. Mix well and incubate for 20 minutes in the dark at four degrees Celsius following the incubation wash and spin the cells as before. Then re suspend the cell pellet in four milliliters of buffer solution For every three mice, place max LS columns in the magnetic field of a suitable max separator.
Prepare the columns by rinsing with four liters of buffer solution. Then apply cell suspension and collect the unlabeled cells that pass through in a collection tube. Wash the column with formula liters of buffer solution three times.
Add new buffer when the column reservoir is emptied. Once all of the solution has flowed through discard flow through, remove the columns from the separator and place each one on top of a 15 milliliter conical tube. Pipette four milliliters of buffer solution onto each column immediately flush out the magnetically labeled cells by pushing the plunger into the column.
Centrifuge the tubes at 500 times G for five minutes. Carefully remove the SUP natum by aspiration. Resuspend each pellet with one milliliter of buffer solution and group pellets in a single 15 milliliter conical tube.
Once the sea kit positive cell population has been enriched, stain and sort the cells by facts. Hematopoietic progenitors and hematopoietic stem cells from a bone marrow suspension enriched for sea kit positive cells using magnetic beads and LT HSC and THSC was sorted according to expression of surface molecules as described in this video. This figure shows an example of the electronic gating procedure to score common lymphoid progenitors as linin.
See it low interleukin seven R positive cells. Lin minus see it positive scar one positive and linin see kit positive scar one. Low cells from the SEE kit positive gait.
Common myeloid progenitors are identified as FC gamma R.Low CD 34 positive cells. Also from the C kit positive gait granulocyte monocyte progenitors are identified as FC gamma are high CD 34 positive cells and megakaryocyte erythrocyte progenitors are identified as FC gamma are low CD 34 negative cells from the LKS eight L-T-H-S-C and S-T-H-S-C are identified as CD 34 negative and CD 34 positive cells respectively. Thus, two enriched and highly purified populations are obtained that are physiologically present in low number to address the impact of autoimmunity on hematopoesis HSCs from control and mice with inflammatory bowel disease.
Were stained with KI 67 antibody and DPI as can be seen here. Cycling cells are barely, if at all, detectable in the LKS CD 34 negative hematopoietic stem cells compartment of healthy controls. In contrast, the same cell subsets is significantly increased during IBD and a substantial number of HSCs are detected in the SG two M phases of the cell cycle.
Thus suggesting that T-cell mediated chronic inflammation determines entry of HSCs into the cell cycle. To assess the presence of a TP stored in vesicles CD 34 negative cells from healthy mice was stained with the nucleotide binding compound quinine and nuclear red analysis by live cell confocal microscopy reveals that in HSCs but not GMPs, quire positive vesicles are visible in the cytoplasm to determine whether HSCs are responsive to extracellular A TP.The calcium response of sorted hematopoietic stem cells loaded with 4 0 2 and stimulated with a TP and ion mycin was analyzed as seen here. Single cell calcium elevation traces indicate that there was an increase in cytosolic calcium in LT HSC by activation of pure anergic P two receptors.
Upon exposure to a TP since cytosolic calcium elevations induce release of vesicular A TP.This experiment suggests that a TP may regulate its own release from HSCs and demonstrates that this procedure can be used for functional studies of HSCs. After watching this video, you should have a good understanding of how to isolate neurone topo stem cells and lineage committed progenitors for functional assays. Following this procedure, other methods like reconstitution of non eradiated recipient mice can be performed in order to answer additional questions like the reconstitution potential of hematopoietic stem cells purified from C State or inflammatory environment.
