Easysep mouse hematopoietic progenitor cell isolation kit

Manufactured by STEMCELL

Sourced in Canada

The EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit is a laboratory tool used to isolate hematopoietic progenitor cells from mouse cell samples. It utilizes magnetic particles and a specialized buffer system to selectively enrich for the target cell population.

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EasySep kits (21 citations) Isolation kits (7 citations) EasySep cell isolation kit (6 citations) EasySep isolation kits (5 citations) Mouse hematopoietic progenitor cell isolation kit (4 citations) Cell isolation kits (4 citations) EasySep Hematopoietic Progenitor Cell Isolation Kit (2 citations) EasySep Mouse Hematopoietic Cell Isolation Kit (2 citations)

43 protocols using easysep mouse hematopoietic progenitor cell isolation kit

Characterizing HSPC Phosphorylation Patterns

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BM cells were first enriched for HSPCs using EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (catalog, 19856, Stem Cell Technologies, Vancouver, BC), and then stained with surface antibodies, after which stained cells were fixed and permeabilized as described above before staining with anti-phospho-MLKL (Abcam, Cambridge, UK). Finally, cells were stained with DAPI (Life Technologies), and image acquisition was performed with Amnis ImageStream Mark II imaging flow cytometer (Luminex, Austin, TX). Raw data were analyzed with IDEAS v6.1 software for co-localization.

Nguyen T.H., Abidin B.M, & Heinonen K.M. (2022). Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis. Stem Cell Reports, 17(10), 2303-2317.

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Lentiviral Transduction of Murine Hematopoietic Progenitors

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For all studies, Lin- cells were enriched from total BM isolated from femurs and tibias of donor mice (6–12 weeks of age) using the EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (STEMCELL Technologies) in conjunction with the RoboSep-S (STEMCELL Technologies) automated cell-isolation machine and confirmed by flow analysis. Cells were stained for lineage markers with a PE-Cy5-conjugated lineage marker cocktail including B220, Ter119, Tcrb, Cd8a, Cd3ε, Cd4, Ly6g/Ly6c, and Cd11b and with hematopoietic stem cell markers APC-conjugated anti-Cd117 (c-Kit) and PE-conjugated Ly6a/e (Sca-1) (BD Biosciences). Lin- cells were transduced with a lentiviral vector in a cell incubator at 2 × 10⁶ cells/mL in either StemSpan SFEM II (STEMCELL Technologies) or StemMACS HSC Expansion Media (Miltenyi Biotec) growth media freshly supplemented with TPO (10 ng/mL), SCF (100 ng/mL), and FLT3 (50 ng/mL). Approximately 16–22 h later, cells were collected, washed at least three times with DPBS (without Ca²⁺ and Mg²⁺), and resuspended in an appropriate volume for dosing. A small number of cells (3,000–4,000 cells) were set aside for use in the colony-forming unit assay using M3434 media (STEMCELL Technologies) and were analyzed 7–10 days later.

Plasschaert R.N., DeAndrade M.P., Hull F., Nguyen Q., Peterson T., Yan A., Loperfido M., Baricordi C., Barbarossa L., Yoon J.K., Dogan Y., Unnisa Z., Schindler J.W., van Til N.P., Biasco L, & Mason C. (2022). High-throughput analysis of hematopoietic stem cell engraftment after intravenous and intracerebroventricular dosing. Molecular Therapy, 30(10), 3209-3225.

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Mobilizing Hematopoietic Progenitor Cells

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Wild-type mice were given 0.2 mL of 50 μM ergosterol i.v. or equal v/v vehicle. Twenty-four hours later, mice received AMD3100 (3 mg/kg) via tail vein injection. One hour after AMD3100 administration, mice were killed via carbon dioxide asphyxiation and whole blood assessed for total cell counts on a Hemavet 950FS Analyzer. To analyze CFU-C, whole blood was first lineage depleted using the EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit according to the manufacturer's instructions (STEMCELL Technologies). To perform CFU-C assays, lineage-negative cells were cultured on MethoCult GF M3434 (STEMCELL Technologies) according to the manufacturer's instructions and readout after 14 days.

Astuti Y., Kramer A.C., Blake A.L., Blazar B.R., Tolar J., Taisto M.E, & Lund T.C. (2016). A Functional Bioluminescent Zebrafish Screen for Enhancing Hematopoietic Cell Homing. Stem Cell Reports, 8(1), 177-190.

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Isolation of Hematopoietic Stem and Progenitor Cells

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HSPCs were isolated using EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (STEMCELL Tech, 19856 A) according to the manufacturer’s protocol. Briefly, BM cells from Apoe^−/−, Apoe^−/−Il27ra^+/−, and Apoe^−/−Il27ra^−/− mice were incubated for 15 min at 4 °C with hematopoietic progenitor cells biotin isolation cocktail followed by incubation with Streptavidin RapidSpheres for 10 min at 4 °C. Tubes with cell suspension were placed into the magnet and incubated for 3 min followed by pouring the enriched cell suspension. Collected fraction represented the enriched lineage negative cell fraction (HSPCs). After isolation, HSPCs were counted and used for gene expression, western blotting, or colony-formation assays.

Peshkova I.O., Aghayev T., Fatkhullina A.R., Makhov P., Titerina E.K., Eguchi S., Tan Y.F., Kossenkov A.V., Khoreva M.V., Gankovskaya L.V., Sykes S.M, & Koltsova E.K. (2019). IL-27 receptor-regulated stress myelopoiesis drives abdominal aortic aneurysm development. Nature Communications, 10, 5046.

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Isolation and Characterization of Murine Hematopoietic Stem Cells

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All animal work in this study was carried out in accordance with regulations set by the United Kingdom Home Office and the Animal Scientific Procedures Act of 1986. Bone marrow was isolated from the spine, femora, tibiae, and ilia of 8 weeks and 72 weeks old C57BL/6J mice. Red blood cell depletion was performed with ammonium chloride lysis (STEMCELL Technologies), and lineage-negative cells were isolated using the EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (STEMCELL Technologies).
The lineage-depleted cells were stained with the following fluorophore-conjugated monoclonal antibodies: Cd105-PE, clone MJ7/18, Miltenyi; Cd4-Vioblue, clone REA604, Miltenyi; Cd11b-Vioblue, clone REA592, Miltenyi; Cd117-Pe Vio770, clone REA791, Miltenyi; Cd8a-Vioblue, clone 53-6.7, Miltenyi; Cd50-Vioblue, clone REA421, Miltenyi; Cd45R-Vioblue, clone REA755, Miltenyi; GR1-Vioblue, clone REA810, Miltenyi; Sca-APC, clone REA422, Miltenyi; Cd48-APC Cy7, clone HM48-1, Miltenyi; Cd150-BV510, clone TC15-12F12, Cd34-PeCy5, MEC147, Miltenyi. Approximately 10,000 LK (Lin−, Cd117+) cells per sample were sorted using the BD FACSMelody cell sorter (BD Biosciences, San Jose, California) into 1× PBS containing 4% BSA. For low-input and single-cell qPCR, a pool of 25 cells and single LSK Cd150+ Cd48− Cd34− HSCs respectively were sorted directly into Smart-seq2 lysis buffer^{51 (link)}.

Mincarelli L., Uzun V., Wright D., Scoones A., Rushworth S.A., Haerty W, & Macaulay I.C. (2023). Single-cell gene and isoform expression analysis reveals signatures of ageing in haematopoietic stem and progenitor cells. Communications Biology, 6, 558.

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LSK Cell Expansion by VTP50469 Treatment

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Hematopoietic stem cell-enriched Lineage⁻ Sca1⁺c-KIT/CD117⁺ (LSK) cells were isolated from the BM of 8-10 week old C57BL/6 mice (Charles River Laboratories). Lineage depletion was performed using EasySep™ Mouse Hematopoietic Progenitor Cell Isolation Kit (STEMCELL Technologies). Subsequently, cells were washed, and lin⁺ cells were removed using magnetic beads (Dynabeads; Invitrogen). Cells were identified with anti-mouse Sca1-Alexa Fluor 647 and anti-mouse CD117-APC (Biolegend) using flow-sorting. LSK cells (500 cells/plate in triplicate) were cultured in StemSpan SFEM (STEMCELL Technologies) containing 100 ng/ml rmSCF/rmFLT3/rmTPO with VTP50469 (30nM) or DMSO (0.1%) for two rounds of replating. At each replating, the number of colonies was scored.

Krivtsov A.V., Evans K., Gadrey J.Y., Eschle B.K., Hatton C., Uckelmann H.J., Ross K.N., Perner F., Olsen S.N., Pritchard T., McDermott L., Jones C.D., Jing D., Braytee A., Chacon D., Earley E., McKeever B.M., Claremon D., Gifford A.J., Lee H.J., Teicher B.A., Pimanda J.E., Beck D., Perry J.A., Smith M.A., McGeehan G.M., Lock R.B, & Armstrong S.A. (2019). A Menin-MLL inhibitor induces specific chromatin changes and eradicates disease in models of MLL-rearranged leukemia. Cancer cell, 36(6), 660-673.e11.

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Multiparameter Flow Cytometry Assay

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Cells were stained with the indicated fluorophore-conjugated antibody or dye according to manufacturer’s recommendations. Antibodies used in flow cytometry experiments can be found in Supplemental Table 1. Prior to flow cytometry analysis, mouse peripheral blood was incubated in ACK buffer (Gibco) and bone marrow was lineage depleted with the EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (StemCell Technologies) according to manufacturer protocols. Flow cytometry analysis or sorting was performed on a BD LSRII or FACSAria (BD Biosciences), respectively. Data was analyzed in FlowJo v.10.0.8 (FlowJo, LLC, Ashland, OR).

Richter L.E., Wang Y., Becker M.E., Coburn R.A., Williams J.T., Amador C, & Hyde R.K. (2019). HDAC1 is a Required Cofactor of CBFβ-SMMHC and a Potential Therapeutic Target in Inversion 16 Acute Myeloid Leukemia. Molecular cancer research : MCR, 17(6), 1241-1252.

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Isolation and Purification of HSCs

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BM was first lineage-depleted using EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (StemCell Technologies, Vancouver, Canada), then stained for HSC markers as above, here including Lin, cKit, Sca1, and CD150. Cell sorting was performed using a SORP Aria flow cytometer (BD Biosciences). HSCs were sorted directly into lysis buffer for total RNA extraction using the RNeasy Plus Micro kit (Qiagen, Hilden, Germany).

Patterson A.M., Vemula S., Plett P.A., Sampson C.H., Chua H.L., Fisher A., Wu T., Sellamuthu R., Feng H., Katz B.P., DesRosiers C.M., Pelus L.M., Cox G.N., MacVittie T.J, & Orschell C.M. (2022). Age and Sex Divergence in Hematopoietic Radiosensitivity in Aged Mouse Models of the Hematopoietic Acute Radiation Syndrome. Radiation research, 198(3), 221-242.

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Isolating Hematopoietic Stem Cells from Aged Mice

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Bone marrow cells from young and old adult mice were aseptically collected from the non-injured lower extremity (femur bone) 1 day after polytrauma (old PT n=8, young PT n=8) or 1 day after pneumonia (old PT+PNA n=7, young PT+PNA n=7), as well as from naïve young and old adult control mice. Single cell suspensions were created by passing the cells through 70 μm pore sized cell strainers (BD Falcon, Durham, NC). Erythrocytes were lysed using ammonium chloride lysis buffer and washed with phosphate-buffered saline. HSPCs were isolated via immunomagnetic negative selection using EasySep™ Mouse Hematopoietic Progenitor Cell Isolation kit (StemCell Technologies, Vancouver, BC) according to the manufacturer’s protocol (9 (link)).

Darden D.B., Mira J.C., Lopez M.C., Stortz J.A., Fenner B.P., Kelly L.S., Nacionales D.C., Budharaju A., Loftus T.J., Baker H.V., Moore F.A., Brakenridge S.C., Moldawer L.L., Mohr A.M, & Efron P.A. (2021). Identification of Unique microRNA Expression Patterns in Bone Marrow Hematopoietic Stem and Progenitor Cells after Hemorrhagic Shock and Polytrauma in Young and Old Adult Mice. The journal of trauma and acute care surgery, 91(4), 692-699.

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Isolation of mouse hematopoietic progenitor cells

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Mouse bone marrow cells were collected from the femurs, tibiae and iliac crest and depleted of red blood cells by an ammonium chloride lysis step (STEMCELL Technologies). Cells were lineage depleted using EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (19856, STEMCELL Technologies). Lineage⁻ c-Kit⁺ (LK) cells were then isolated using the following antibodies (clone and company): streptavidin BV510 (BioLegend), c-kit APC-Cy7 (2B8, BioLegend), Sca1 BV421 (D7, BioLegend), CD45 FITC (30-F11, BioLegend), EPCR (CD201) PE (RMEPCR1560, STEMCELL Tech), CD150 PE/Cy7 (TC15-12F12.2, BioLegend), CD45 FITC (30-F1,1 BD Bioscience) and CD48 APC (HM48-1, eBioscience). Flow cytometry was performed on an LSRII Fortessa (BD) and all data were analyzed using FlowJo (BD). A representative gating strategy is shown in Figure S12.

Isobe T., Kucinski I., Barile M., Wang X., Hannah R., Bastos H.P., Chabra S., Vijayabaskar M.S., Sturgess K.H., Williams M.J., Giotopoulos G., Marando L., Li J., Rak J., Gozdecka M., Prins D., Shepherd M.S., Watcham S., Green A.R., Kent D.G., Vassiliou G.S., Huntly B.J., Wilson N.K, & Göttgens B. (2023). Preleukemic single-cell landscapes reveal mutation-specific mechanisms and gene programs predictive of AML patient outcomes. Cell Genomics, 3(12), 100426.

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