For granulocyte-macrophage colonies 30 000 fresh BM cells from steady state mice were plated in methylcellulose medium (M3231; StemCell Technologies) containing 50 ng/mL mSCF, 10 ng/mL murine interleukin 3 (IL-3; PreproTech), 10 ng/mL human IL-6 (PreproTech), 100 IU penicillin and 100 μg streptomycin (P/S; Invitrogen) in 35-mm petri dishes. For erythroid colonies 150 000 BM cells were plated in methylcellulose medium (M3236; StemCell Technologies), supplemented with 50 ng/mL mSCF, 50 ng/mL hTPO and 5 U/mL human erythropoietin (Epo; Apoteket Farmaci) and P/S (Invitrogen). Colony numbers were scored after 12 days of culture. For total colony capacity of cultured CD34+ human cells, 250–300 cells were plated in 35-mm petri dishes in methylcellulose medium (M4230; StemCell Technologies) containing hSCF (25 ng/mL), GM-CSF (50 ng/mL, R&D), hIL-3 (25 ng/mL, PreproTech), hEPO (5 U/mL, Apoteket Farmaci, Lund, Sweden), and P/S (Invitrogen). Total colony number was scored after 12–14 days of culture. Cells were incubated at 37 °C in 5% CO2.
M3236
Manufactured by STEMCELL
Sourced in Canada
The M3236 is a laboratory equipment product designed for cell culture applications. It serves as a mechanical and thermal incubation system to maintain appropriate conditions for cell growth and experimentation. The core function of the M3236 is to provide controlled temperature and atmospheric conditions within a sealed chamber.
Automatically generated - may contain errors
Lab products found in correlation
Heparin, by Merck Group (4 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (3 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (3 mentions)
Interleukin 3 (il 3), by R&D Systems (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Epidermal growth factor (egf), by R&D Systems (2 mentions)
Insulin like growth factor 1, by R&D Systems (2 mentions)
Fitcbs lectin 1, by Vector Laboratories (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dii ldl, by Biomedical Technologies (2 mentions)
Paraformaldehyde pfa, by Merck Group (2 mentions)
Human il 6, by Thermo Fisher Scientific (1 mentions)
Hil 3, by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
M3231, by STEMCELL (1 mentions)
Gm csf, by R&D Systems (1 mentions)
Interleukin 3 il 3, by R&D Systems (1 mentions)
Basic fibroblast growth factor (bfgf), by STEMCELL (1 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (1 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
6 protocols using m3236
1
Quantifying Hematopoietic Progenitor Colonies
2
Quantification of Hematopoietic Progenitor Colonies
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After isolation of c-kit+/Sca-1−/Lin− (KSL) cells from mouse BM (Kwon et al., 2011 (link)), the frequencies of small or large colonies was determined after culturing KSL cells (500 cells/35 mm dish) for 10 days in methylcellulose-containing medium M3236 (Stem Cell Technologies) supplemented with 20 ng/ml stem cell factor (SCF), 50 ng/ml VEGF, 20 ng/ml interleukin-3 (IL-3) (R&D Systems), 50 ng/ml bFGF, 50 ng/ml EGF (PeproTech), 2 U/ml heparin (Sigma), 30% FBS, and antibiotics. Colony forming units (CFUs) were counted under an inverted microscope at 40x. In our previous study, we defined small- and large-CFUs for the expression of additional endothelial marker genes such as CD31, Flk-1, von Willebrand factor (vWF), VE-cadherin and eNOS (Kwon et al., 2011 (link)).
3
Quantifying Endothelial Progenitor Cells
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In order to evaluate the EPC colony forming unit (EPC-CFU), 1 × 106 peripheral blood mononuclear cells of each group of mice were cultured in methylcellulose-containing medium M3236 (StemCell Technologies). Stem cell-derived factors are 20 ng/mL, 50 ng/mL vascular endothelial growth factor, 20 ng/mL IL-3, 50 ng/mL basic fibroblast growth factor, 50 ng/mL epidermal growth factor, 50 ng/mL insulin-like growth factor-1 (all from R&D Systems), 2 U/mL heparin (Sigma-Aldrich), and 10% FBS (Gibco), which were added. After culturing for 10–12 days, the EPC-CFU culture was treated with 0.4 mg/mL DiI-LDL (Biomedical Technologies) for 2 hours and fixed by applying 1 mL of 2% paraformaldehyde (PFA, Sigma-Aldrich) for 1 hour at room temperature. After washing the methylcellulose-containing medium with PBS, the culture was reacted with FITCBS-lectin 1 (Vector Laboratories) at room temperature for 1 hour. After washing with PBS, the culture was observed under a fluorescence microscope (IX70; Olympus). The number of EPC-CFU reflects the number of original EPCs in the initial classified cell fraction.
4
Endothelial Progenitor Cell Culture and Characterization
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Peripheral blood‐derived MNCs (2 × 105 cells) were cultured for 12 days in methyl cellulose‐containing medium M3236 (Stemcell Technologies, Vancouver, Canada; Center Valley, PA, www.olypusamerica.com ) containing 20 ng/ml of stem cell factor, 50 ng/ml vascular endothelial growth factor, 20 ng/ml interleukin‐3, 50 ng/ml basic fibroblast growth factor, 50 ng/ml epidermal growth factor receptor, and 50 ng/ml insulin‐like growth factor‐1. All supplementary growth factors were purchased from PeproTech (Rocky Hill, NJ, www.peprotech.com ). The endothelial phenotypes of the colonies were confirmed by high uptake of acetyl LDL (DiI acetylated low‐density lipoprotein [DiI‐Ac‐LDL], Biomedical Technologies, Stoughton, MA, www.btiinc.com ) and cytochemical positivity for isolectin B4 (ILB4; Vector Laboratories, Burlingame, CA, vectorlabs.com ).
5
Endothelial Potential of KSL Cells: Colony Formation Assay
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EPC colony formation was assessed by culturing 500 KSL cells in triplicate in methylcellulose-containing medium M3236 (Stem Cell Technologies) supplemented with; 100 ng/ml SCF (PeproTech), 50 ng/ml VEGF (PeproTech), 20 ng/ml interleukin-3 (R&D Systems), 50 ng/ml basic fibroblast growth factor (bFGF, R&D Systems), 50 ng/ml epidermal growth factor (EGF, PeproTech), 2 U/ml heparin (Sigma Aldrich), 30% FBS, and antibiotics. Small- and large-colonies (CFUs) after culture for 12 days were defined as focused clusters of rounded cells or as cell clusters with a central core of round cells and elongated sprouting cells at their peripheries [51 (link)]. Endothelial characteristics of the attached small- and large-CFUs were examined after uptake of DiI-conjugated Ac-LDL (DiI-Ac-LDL) (Biomedical Technologies Inc., MA) and binding with FITC-conjugated isolectin B4 (Sigma Chemical Co., WI), a standard marker of endothelial lineage cells. In our previous study, we defined these CFUs for the expression of additional marker genes such as CD31, Flk-1, eNOS [7 (link)], and in this study von Wilibrand factor (vWF), and VE-cadherin.
6
Quantifying Endothelial Progenitor Cells
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To evaluate EPC colony forming units (EPC-CFUs) of PB, 1 × 106 PBMNCs from vehicle control or Me6-injected mice were cultured in methylcellulose-containing medium M3236 (StemCell Technologies) with 20 ng/mL stem cell-derived factor, 50 ng/mL vascular endothelial growth factor, 20 ng/mL interleukin-3, 50 ng/mL basic fibroblast growth factor, 50 ng/mL epidermal growth factor, 50 ng/mL insulin-like growth factor-1 (all from R&D Systems), 2 U/mL heparin (Sigma-Aldrich), and 10% FBS (Gibco) for 10–12 days. After 10–12 days in culture, the EPC-CFU cultures were treated with 0.4 μg/mL DiI-LDL (Biomedical Technologies) for 2 hour and fixed by application of 1 mL of 2% paraformaldehyde (PFA, Sigma-Aldrich) for 1 hour at room temperature. After a wash of the methylcellulose-containing medium with PBS, the cultures were reacted with FITC-BS-lectin 1 (Vector Laboratories) for 1 hour at room temperature. After a wash with PBS, the cultures were observed under a fluorescence microscope (IX70; Olympus). The number of EPC-CFUs reflected the number of primitive EPCs in the initial sorted cell fractions38 (link).
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