RBCs from PB were lysed with BD Pharm Lyse buffer (BD Biosciences). Nucleated cells were subsequently washed twice and used for CFU-GM colonies. Briefly, cells were resuspended in human methylcellulose base media provided by the manufacturer (R&D Systems, Inc., Minneapolis, MN, USA), supplemented with 25 ng/ml recombinant murine granulocyte macrophage colony-stimulating factor (mGM-CSF) and 10 ng/ml recombinant murine interleukin 3 (mIL-3; Millipore, Billerica, MA, USA). Cultures were incubated for 7 days, at which time they were scored for the number of CFU-GM colonies under an inverted microscope. To evaluate the number of clonogenic progenitor cells, BMMNCs were supplemented with erythropoietin (5 U/ml; Stem Cell Tech., Vancouver, BC, Canada) plus stem cell factor (5 ng/ml) and resuspended in methylcellulose base medium (for determining the number of burst-forming units-erythroid), supplemented with thrombopoietin (100 ng/ml) plus murine interleukin 3 (10 ng/ml), and the resuspended plasma clots used for determining the number of CFU-megakaryocytes. The CFU-GM assay was performed as above. Cultures were incubated for 7 days (37°C, 95% humidity, and 5% CO2), at which time they were scored under an inverted microscope for the number of each type of colony, as described17 (link),19 (link).
Human methylcellulose base media
Manufactured by R&D Systems
Sourced in United States
Human methylcellulose base media is a cell culture medium designed for the growth and maintenance of human cells. It provides a semi-solid matrix that supports the formation of 3D cellular structures, such as spheroids and organoids. The medium is formulated to support the survival and proliferation of a variety of human cell types, including stem cells, primary cells, and established cell lines.
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Lab products found in correlation
6 protocols using human methylcellulose base media
1
Quantification of Hematopoietic Progenitor Cells
2
Clonogenic Potential of DJ4 in Methylcellulose
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The cells were cultured at a constant optimal seeding density in Human Methylcellulose Base Media (R&D Systems, Minneapolis, MN, USA) in a 12-well plate to yield colony outgrowth of 20–100 colonies per well as described previously [51 (link),52 (link)]. To test the clonogenic potential, the cells were cultured in the presence of increasing concentrations of DJ4 (0–10 μM) or the DMSO vehicle in a methylcellulose medium for 7–14 days. Blast colonies (>20 cells/colony) were counted under a light microscope and imaged with an Olympus CKX31 inverted microscope (Olympus Corporation, Center Valley, PA, USA) using a 4× objective.
3
Quantifying Hematopoietic Colony Formation
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For human cell lines, 5 × 103 cells were resuspended in Human Methylcellulose Base Media (R&D) and plated in 35-mm culture dishes. Cells were allowed to grow for 7–10 days and then colonies were pooled, counted, and 5 × 103 cells were re-seeded for re-plating experiments. When indicated Zn (100 μM) was added to the methylcellulose medium. In pre-treatment experiments (Fig 4 B), NB4 cells were treated in liquid medium with ATRA (1 μM) for 24 h and plated in methylcellulose after drugs washout. In CFU-L experiments of BM cells from leukemic mice, 3 × 104 cells were seeded in MethoCult® GF Medium (STEMCELL Technologies) 24 h after transfection with EZN-3088 and EZN-2968, and colonies were counted 7–10 days after plating.
4
Quantifying CFU-GM Colonies from PBMNC
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Red blood cells (RBCs) were lysed with BD Pharm Lyse buffer (BD Biosciences, San Jose, CA). Nucleated cells from PB were subsequently washed twice and used for CFU-GM colonies, cells were resuspended in human methylcellulose base media provided by the manufacturer (R&D Systems, Inc., Minneapolis, MN) supplemented with 25 ng/ml recombinant murine granulocyte macrophage colony-stimulating factor (mGM-CSF) and 10 ng/ml recombinant murine interleukin-3 (mIL-3; Millipore, Billerica, MA). Cultures were incubated for 7 days, at which time they were scored for the number of CFU-GM colonies under an inverted microscope as described [8 –10 (link)]. We cultured 1 × 106 PBMNC/dish and final results are recalculated based on number of PBMNC/1 μl of peripheral blood.
5
Murine Macrophage Colony Assay
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Red blood cells were lysed with BD Pharm Lyse buffer. Nucleated cells were subsequently washed twice and used for CFU-M colonies. Bone marrow nucleated cells (BMNCs) were resuspended in human methylcellulose base media provided by the manufacturer (R&D Systems, Minneapolis, MN, USA) supplemented with 10 ng/mL recombinant murine macrophage stimulating factor (mM-CSF) for CFU-M. Cultures were incubated for 7 days, at which time they were scored for the number of CFU-M colonies under an inverted microscope.
6
Clonogenic Potential of AML Cells
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Cryopreserved human AML patient or cord blood mononuclear cells were cultured in Human Methylcellulose Complete Media (R&D Systems, Minneapolis, MN) in triplicate in 12-well plates at a density of 0.1–2 × 105 cells per well. The optimal plating densities were selected for each case to yield colony out-growth of 20–100 colonies per well. Human AML cell lines were cultured at a density of 250–500 cells per well in Human Methylcellulose Base Media (R&D Systems). Two approaches were used to test the clonogenic potential of cells treated with ISC-4 against clonogenicity, first, cells were cultured in the presence of increasing concentrations of ISC-4 or DMSO simultaneously in methylcellulose medium for 7–14 days. Second, cells were exposed to ISC-4 (1–10 μM) or DMSO for 24 h. Post-treatment, cells were washed, counted and plated in methylcellulose-containing medium to grow colonies. Colonies were propagated for 7–14 days, and blast colonies (>20 cells/colony) were counted in a blinded manner under the light microscope. Colonies were imaged with the Olumpys CKX31 inverted microscope (Olympus corporation, Center Valley, PA) using the 4X objective.
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