Lo Celso, C., Scadden, D. Isolation and Transplantation of Hematopoietic Stem Cells (HSCs). If you would like to continue using JoVE, please let your librarian know as they consider the most appropriate subscription options for your institution’s academic community. Wash and resuspend the cells into 100 ml of 2%BSA/PBS and add appropriate amount of antibody, mix by pipetting and incubate on ice for 10 minutes in dark. The retronectin coating of the cell culture plastic surfaces enables the adhesion of primitive HSCs to the dish and increases the virus-mediated transduction of the cells (9). Mouse hematopoietic stem cells (HSCs) are the best-studied stem cells because functional assays for mouse HSCs were established earliest and purification techniques for … Carefully pipet off “cloudy” interface layer (~10 mL) and transfer into a clean 50-mL tube. If you do not receive an email within 10 minutes, your email address may not be registered, During the incubation, prepare the columns on the magnets and 15ml elution tubes under the columns. Divide the cells equally to three tubes, add 2 ml of antibodies (Ly5.2 FITC, Ly5.1 PE, and isotype control), and incubate on ice for 10 minutes. J. Vis. At the end of the incubation, add PBS 2% FCS to fill the tubes and spin down for 5 min at 1200rpm. Copyright © 2020 MyJoVE Corporation. However, HSCs can be cultured in an undifferentiated state for a few days on retronectin pretreated cell culture dishes in high quality medium supplemented with fetal bovine serum (FBS), penisillium-streptomycin, and cytokines. Godin, I. E., Garcia-Porrero, J. This is a preview of subscription content.
The preparation details vary, depending on the specific tissue source. 2494-2499. The bone marrow cells are then centrifuged for 5 minutes with 1500 rpm, the cells are resuspended into a maximum of 1 ml of DMEM, and loaded carefully on the gradient (500 ml Ficoll in 1.8 ml eppendorf tube since the use of small eppendorf tubes instead of 15 ml conical tubes improves the yield of the isolation.). The pellet should be white or mainly white. pp 1-14 | Figure 1. Muller, A. M., Medvinsky, A., Strouboulis, J., Grosveld, F., and Dzierzak, E. (1994) Development of hematopoietic stem cell activity in the mouse embryo. Even though mouse hematopoietic system closely resembles human hematopoietic compartment to date the above-mentioned classification apply only to mouse models. Thank you for taking us up on our offer of free access to JoVE Education until June 15th. It might take a while to get the cells to go through these filters, and it is important to apply some more PBS afterwards to wash the filter and minimize cell loss.
Colony formation unit (CFU) cell assay results of human CD34+ hematopoietic progenitor cells. The dishes can be stored at +4 °C tightly wrapped in foil. Cut the skin quickly with the scissors to expose the limbs and remove the muscles surrounding the bones (Figure 1B-F). Exp. Therefore they are considered as the ideal targets for various clinical applications including stem cell transplantation and gene … If the irradiation is given in one single dose, the maximum is 850 cGy. However, the dose of irradiation could be different for different strains of the mice and therefore should be optimized depending on the animal model used. Once the columns are empty, apply again using the same 40μm filter. https://doi.org/10.1385/1-59259-140-X:001. This step is necessary to avoid clogging of the sorter, which is the worst nightmare of every cell sorter user.
Hanenberg et al. Mice: 4-8 weeks old donor and 6-9 weeks old recipient mice. At this stage, the filter is used to separate bone debris. The cell mixture to sort is stained in the 15ml tube. 870 Downloads; Part of the Methods in Molecular Medicine book series (MIMM, volume 63) Abstract. Expansion of Human CD34+ Hematopoietic Progenitor Cells The edges of the bone are cut gradually to expose the bone cavity. Loosen the pellets and resuspend them together in 1ml total PBS 2% FCS if you are sorting later, otherwise resuspend in whatever volume is convenient for you. Accelerate Your Research, Education, and Learning. The retroviral or lentiviral manipulation of the cells should be done on three consecutive days with a fresh high titer virus.
2010 Nov;50(11):2402-12. doi: 10.1111/j.1537-2995.2010.02743.x. The donor mouse is sacrificed humanely according to established animal care protocols approved by animal care committees and regulatory agencies, rinsed with 70% ethanol, and moved to laminar hood for bone marrow isolation from both hind limb tibias and femurs (Figure 1A). 450 cGy for 2 minutes, Let the mice to rest for 3-6 hours before the second irradiation dose. Leave mice to rest in their cages for 8-18 hours to reduce the cytokine storm and increased apoptotic response caused by the TBI before injecting the graft. Keywords: Resuspend each tube in 1 ml PBS 2% FCS (NO EDTA from now on) if you are doing lineage depletion, or in whatever volume is otherwise convenient for you. Cohen etl al. During the dissections, the limbs and the bones are kept in PBS 2%FCS heat inactivated (optional 2mM EDA - dissection medium).
A commonly used cytokine combination include stem cell factor (SCF, steel factor, c-Kit ligand), which is needed to maintain the stemness of HSCs, throbopoietin (TPO) and Flt3 ligand, which both are promoting the cell proliferation (10,11,12). Once the femur and the tibia are separated carefully cut the ends of the bones close to the joints by scissors to open the bone marrow cavity (Figure 1I). Subsequently puncture the bone marrow cavity carefully with a needle. Human hematopoietic stem cell markers analyzed by flow cytometry. The eluate contains the lineage depleted cell mixture, a heterogeneous population enriched for stem and progenitors cells. If you are sorting HSC, this step drastically reduces the number of cells that have to go through the cell sorting, therefore reducing sorting time and allowing you to sort HSC out of a higher number of mice, so that you can obtain a higher number of HSC in one single sorting. We may use this info to send you notifications about your account, your institutional access, and/or other related products.
The cells are injected in 200 ml of PBS into the tail vein using butterfly needle (30G). Count the cells and use minimum of 1x105 cells for the staining. In this way, each tube contains the cell mixture obtained out of all the leg bones of a mouse, and half of the mixture obtained from 2 spines. 2007;368:237-59. doi: 10.1007/978-1-59745-362-2_17. Wash cells twice and resuspend in Hank’s +, Before the fluidic system is turned on, remove the fluid filter and use a syringe and blunt needle to inject a sufficient volume of filtered 10% bleach (. The cell mixture obtained from each mouse is kept separate and filtered through a 40μm filter into a 50ml falcon tube. Aspirate the supernatant, loosen the pellets and resuspend in 1 ml/tube degassed PBS. Hogan, B., Costantini, F., and Lacy, E. (1986).
Exp. Spangrude, G. J., Heimfeld, S., and Weissman, I. L. (1988) Purification and characterization of mouse hematopoietic stem cells. Resuspend the cells in PromoCell HPCs Expansion Medium DXF (, Thaw a vial of frozen CD34+ hematopoietic stem cells (, Suspend the cells and transfer the entire 0.3 ml cell suspension to a 3 ml aliquot of thawed and supplemented Hematopoietic Progenitor Expansion Medium DXF (. Jordan, C. T. and Lemischka, I. R. (1990) Clonal and systemic analysis of longterm hematopoiesis in the mouse. Since the irradiation is damaging to the intestinal tissues the mice become sensitive to internal infections. Springer Nature is developing a new tool to find and evaluate Protocols. Blood 90: 4384-4393. If you want more info regarding data storage, please contact gdpr@jove.com. Protoc. Lo Celso, C., Scadden, D. Isolation and Transplantation of Hematopoietic Stem Cells (HSCs). Spin for 5 min at 1500rpm. (2003). Blood 77: 2316-2321. Clinical transplantation studies that used enriched CD34+ cells from bone-marrow, umbilical cord and peripheral blood mononuclear cells (PBMCs) indicated the presence of HSCs with long-term tissue reconstitution ability. Alternatively, the intestinal pH of the mice can be decreased to protect the animals against microbes by giving autoclaved acidified water (pH 3.0) for two weeks prior the irradiation, which should be continued for two weeks after the irradiation. Molecular cloning of a ligand for the flt3/flk-2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells. Hematopoietic stem cells (HSCs) are multipotent cells capable of differentiating into all types of blood cells. Take out lineage depleted single color staining ctrl (15ml).
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