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8 protocols using methocult gf m3534 medium

1

BM Cell Colony Forming Assay

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Thirty thousand BM cells were cultured per 30 mm dish in MethoCult™ GF M3534 medium following the manufacturer’s protocol (Stem Cell Technologies, Vancouver, BC, Canada) and granulocyte-macrophage colony-forming units (CFU-GM) were counted under a microscope following culture for 9 days.
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2

Bone Marrow Hematopoietic Progenitor Assay

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Granulocyte-macrophage colony-forming unit assays of freshly sorted bone marrow linc-kit+ cells were performed by culturing the cells in Methocult GF M3534 medium (StemCell Technologies, Vancouver, BC, Canada) in the absence and presence of recombinant murine SHH (200 ng/ml, eBioscience/Thermo Fisher Scientific). One milliliter of Methocult GF M3534 medium containing 100 cells was plated to a 35 mm NunclonTM dish (Nunc, Rodkilde, Denmark). The cultures were conducted for seven days at 37°C in an atmosphere of 5% CO2. Colonies containing 50 or more cells were then enumerated.
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3

Hematopoietic Stem Cell Assays

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500 YFP+ LICs were sorted into 1-mL MethoCult™ GF M3534 medium (STEMCELL Technologies) supplemented with growth factors IL-2, IL-6 and SCF (PeproTech), mixed well and plated into one well of a 12-well plate. Colony numbers were determined on day 6. Mouse bone marrow cells were plated in M3534 medium supplemented with IL-3, IL-6, SCF, Epo and indicated concentration of WNT inhibitors. Colonies were scored on day 3 for CFU-E (colony-forming unit erythroid) and on day 7 for BFU-E (burst-forming unit erythroid), CFU-Mk (megakaryocyte) and myeloid colonies including CFU-G (granulocyte), CFU-M (macrophage) and CFU-GM. Experiments were in triplicate.
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4

Quantifying CFU-GM Colonies in Murine Bone Marrow

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Bone marrow cells (2×104) from WT or CGD mice were seeded in semisolid Methocult GF M3534 medium containing rmSCF, rmIL-3 and rhIL-6 for detection of CFU-GM (Stem Cell Technologies). Number of colonies that contained more than 50 cells was counted on day 7. Colony numbers from 20,000 BMMCs are shown. The size of the colonies was also measured on day 7. Colony morphology was scored on the basis of Stemcell Technologies criteria. L-butionine-sulfoxamine (BSO, Sigma-Aldrich) was added to methylcellulose media at the indicated concentrations at the time of plating.
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5

Quantifying Colony-Forming Units from Bone Marrow and Spleen

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Viable bone marrow cells and splenocytes were homogeneously dispersed in the MethoCult™ GF M3534 medium (Stemcell Technologies) at densities of 1 × 104/mL and 2 × 104/mL, respectively. Cells were plated in 35 mm culture dishes (Stemcell Technologies) and incubated at 37°C in a humidified atmosphere of 5% CO2. Following a 9-day (bone marrow cells) and 13-day (splenocytes) incubation, the number of CFU-GM with colonies consisting of more than 50 cells was manually counted under an inverted microscope (BX51, Olympus, Tokyo, Japan). The average colony number of the quadruplicated dishes per group was represented for each specimen.
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6

Murine Hematopoietic Progenitor Assay

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A total of 100 LSKs were seeded in MethoCult GF-M3534 medium (StemCell Technologies) in triplicate in 35-mm plastic plates and cultured at 37°C, 5% CO2, fully humidified air. After 7 days, colony numbers were scored.
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7

Quantification of Mouse Hematopoietic Progenitors

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Detection and quantification of mouse hematopoietic progenitors in peripheral blood was performed using nucleated cells isolated from the pooled peripheral blood of control or 20 mg/kg D16F7 mAb treated B6D2F1 mice (n = 4 for each experimental group) and mixed with MethoCult™ GF M3534 medium (StemCell Technologies, Vancouver, Canada). Briefly, 0.7–0.9 × 106 cells were seeded in 35 mm Petri dishes (8 per group). Cells were allowed to grow for 10 days in CO2 incubator at 37°C and colonies containing at least 30 granulocytes (CFU-G), monocytes/macrophages (CFU-M) or both cell types (CFU-GM) were counted. Results were normalized by total number of initially plated cells.
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8

Multiparametric Flow Cytometry Analysis

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Biotin conjugated anti-mouse-CD4 (clone 34 GK1.5), anti-mouse-CD8 (clone 53-6.7), anti-mouse-CD11b (clone M1/70), anti-mouse-CD45R/B220 (clone RA3-6B2), anti-mouse-Ly6G/Gr-1 (clone RB68C5), anti-mouse-Ter119 (clone Ter119), anti-mouse-CD117 (c-kit)-APC (clone 2B8), anti-mouse -Ly-6A/EA (Sca-1)-PE (clone D7), and PERCP-conjugated streptavidin were purchased from eBioscience (San Diego, CA, USA). In addition, 2,7-dichlorodihydrofluorescein diacetate (DCFDA) was purchased from Sigma (St. Louis, MO, USA). MethoCult GF M3534 medium was purchased from Stem Cell Technologies (Vancouver, Canada). MitSox red mitochondrial superoxide indicator was obtained from Life Technologies (Grand Island, NY, USA). Rabbit anti-γH2AX was obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-NOX4 was obtained from Proteintech (Wuhan, China). FITC-conjugated goat anti-rabbit antibodies were obtained from Abcam Biotechnology (Cambridge, MA, USA). Cytofix/Cytoperm buffer (554722), Perm/Wash buffer (554723), and Cytoperm Permeabilization Buffer Plus (561651) were obtained from BD Pharmingen (San Diego, CA, USA). Sitagliptin was obtained from Merck Sharp & Dohme (South Granville, NSW, Australia).
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