Methocult sfbit

Manufactured by STEMCELL

Sourced in Canada

MethoCult SFBIT is a semi-solid methylcellulose-based medium for the in vitro culture of hematopoietic progenitor cells. It contains recombinant human growth factors and is formulated to support the growth and differentiation of various blood cell lineages from stem and progenitor cells.

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Lab products found in correlation

Eclipse te300, by Nikon (3 mentions) Gridded scoring dish, by STEMCELL (3 mentions) Primaria dishes, by BD (2 mentions) Anti il 10, by Abcam (1 mentions) Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions) Inverted microscope, by Nikon (1 mentions)

4 protocols using methocult sfbit

Angiogenic Potential of PBMNCs in MMD

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To determine the angiogenic potential of PBMNCs, cells derived from patients with MMD (n = 23), patients with MMD-O (n = 7), and controls (n = 23) were cultured in semisolid medium (MethoCult SFBIT; STEMCELL Technologies Inc., Vancouver, British Columbia, Canada) with provasculogenic growth factors/cytokines on 35-mm Primaria dishes (BD Falcon, USA) as reported previously^{10 (link),11 (link)}. EPC colonies that adhered to the dishes were then counted^{9 (link)}. Aliquots of the cells were seeded at 2 × 10⁵ cells/dish (three dishes per PBMNCs/cultured PBMNCs) for the EPC-CFA. The number of adherent colonies per dish was measured using a gridded scoring dish (STEM CELL Technologies, Canada) under a phase-contrast light microscope (Eclipse TE300; Nikon, Tokyo, Japan) 16 to 20 days after the initiation of the culture.
We measured the numbers of small- and large-cell colonies per dish after IL-10 (Pepro Tech, Inc., TX, USA) or anti-IL-10 (Abcam, UK) antibody was added to the freshly isolated PBMNCs or those cultured from patients with MMD (n = 7), patients with MMD-O (n = 3) and controls (n = 6).

Nagata E., Masuda H., Nakayama T., Netsu S., Yuzawa H., Fujii N., Kohara S., Sorimachi T., Osada T., Imazeki R., Matsumae M., Asahara T, & Takizawa S. (2019). Insufficient production of IL-10 from M2 macrophages impairs in vitro endothelial progenitor cell differentiation in patients with Moyamoya disease. Scientific Reports, 9, 16752.

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Vasculogenic Potential of PBMNCs via EPC-CFA

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To investigate the vasculogenic potential of PBMNCs or QQMNCs, we used semisolid culture medium and 35‐mm Primaria dishes (BD Falcon; BD Biosciences) to grow and then counted the adhesive EPC colonies by EPC colony‐forming assay (EPC‐CFA) (MethoCult SF^BIT; STEMCELL Technologies Inc., Vancouver, BC, Canada) with proangiogenic growth factors/cytokines, as previously reported (Table 3).^{12 (link)} Aliquots of those cells were seeded at 2×10⁵ cells/dish (3 dishes per volunteer) for EPC‐CFA. Sixteen to 18 days after initiation of the culture, the number of adherent colonies per dish was measured using a gridded scoring dish (STEMCELL Technologies) under phase‐contrast light microscopy (Eclipse TE300; Nikon, Tokyo, Japan). Primitive EPC colony‐forming units (pEPC‐CFUs) and definitive EPC‐CFUs (dEPC‐CFUs) were separately counted.

Masuda H., Tanaka R., Fujimura S., Ishikawa M., Akimaru H., Shizuno T., Sato A., Okada Y., Iida Y., Itoh J., Itoh Y., Kamiguchi H., Kawamoto A, & Asahara T. (2014). Vasculogenic Conditioning of Peripheral Blood Mononuclear Cells Promotes Endothelial Progenitor Cell Expansion and Phenotype Transition of Anti‐Inflammatory Macrophage and T Lymphocyte to Cells With Regenerative Potential. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 3(3), e000743.

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Endothelial Progenitor Cell Colony Assay

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Fresh PBMNCs derived from healthy volunteers and corresponding QQMNCs were seeded at a concentration of 2 × 10⁵ cells/dish in 35-mm Primaria dishes (BD Falcon) and cultured for 16–18 days using the EPC-CFA system (Methocult SF^BIT; STEMCELL Technologies Inc., Vancouver, BC, Canada). The semi-solid culture medium was supplemented with a growth factor/cytokine mixture of 66.7 ng/mL SCF, 33.3 ng/mL VEGF, 33.3 ng/mL basic fibroblast growth factor (FGF), 33.3 ng/mL EGF, 33.3 ng/mL insulin-like growth factor 1 (IGF-1), 13.3 ng/mL IL-3 (Peprotech, Rocky Hill, NJ, USA), 1.33 IU/mL heparin (Shimizu Pharmaceutical Co., Shizuoka, Japan), and 30% fetal bovine serum (SAFC Biosciences, St. Louis, MO, USA). After 16–18 days in culture, the colonies in each dish were enumerated under an inverted microscope (Nikon Corporation, Tokyo, Japan) at 40× magnification, as described previously^{10 (link)}. Two types of EPC colony-forming units (EPC-CFU) were observed and determined: primitive EPC-CFU (pEPC-CFU) and definitive EPC-CFU (dEPC-CFU)^{5 (link),12 (link),13 (link)}.

Kado M., Tanaka R., Arita K., Okada K., Ito-Hirano R., Fujimura S, & Mizuno H. (2018). Human peripheral blood mononuclear cells enriched in endothelial progenitor cells via quality and quantity controlled culture accelerate vascularization and wound healing in a porcine wound model. Cell Transplantation, 27(7), 1068-1079.

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Quantifying Angiogenic Potential of Blood Cells

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The angiogenic potential of PBMNCs and QQMNCs was assessed using the EPC-CFA, as previously described^{6 (link),14 (link)}. The EPC-CFA was designed to differentiate total EPC colony-forming units (tEPC-CFUs) into two types of EPC-CFUs (primitive and definitive). Primitive EPC-CFU (pEPC-CFUs) are a predominantly proliferative population of cells, and definitive EPCs-CFU (dEPC-CFU) are a predominantly vasculogenic population with greater adhesion, migration, differentiation, and tubularization potential.
Briefly, adhesive EPC colonies were counted using the EPC-CFA (MethoCult SFBIT; Stem Cell Technologies Inc., Vancouver, BC, Canada) with semisolid culture medium containing proangiogenic growth factors/cytokines in 35-mm Primaria dishes (BD Falcon; BD Biosciences). Twelve aliquots of cells were seeded at 2 × 10⁵ cells/dish (three dishes per subject) for EPC-CFA. Sixteen to 18 days after culture initiation, the number of adherent colonies per dish was measured using a gridded scoring dish (Stem Cell Technologies) under phase-contrast light microscopy (Eclipse TE300; Nikon, Tokyo, Japan). The experiments were performed in triplicate. Experimental conditions were masked and two investigators counted the number of pEPC-CFUs and dEPC-CFUs.

Hagiwara H., Higashibata A., Ogawa S., Kanazawa S., Mizuno H, & Tanaka R. (2018). Effectiveness of endothelial progenitor cell culture under microgravity for improved angiogenic potential. Scientific Reports, 8, 14239.

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