Myelocult m5300

Manufactured by STEMCELL

Sourced in Canada

Myelocult M5300 is a serum-free, protein-rich medium designed to support the culture of myeloid progenitor cells, including granulocyte-macrophage progenitor cells (CFU-GM), as well as early and late erythroid progenitor cells (BFU-E, CFU-E). The medium is optimized to maintain the proliferative capacity and differentiation potential of these cells in in vitro culture.

Automatically generated - may contain errors

Lab products found in correlation

21 protocols using myelocult m5300

Clonogenic Assay of Sorted LSK Cells

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Primary bone marrow feeder layers were cultured and established using MyeloCult M5300 and then irradiated according to the protocol from StemCell Technologies. 1 × 10³ sorted LSK cells were cultured on feeder layers at 33° in 5% CO₂ for 4 weeks with a weekly half media change. Cells were then harvested and plated into MethoCult GF M3434 in triplicate. Colonies were determined after 12 days and expressed as number of the CFUs per 1 × 10³ sorted LSK cells as previously reported [14 (link)].

Wu J., Zhang B., Li S., Chen W., Mao J., Li K., Liu D, & Duan Y. (2022). Peptidoglycan-Mediated Bone Marrow Autonomic Neuropathy Impairs Hematopoietic Stem/Progenitor Cells via a NOD1-Dependent Pathway in db/db Mice. Stem Cells International, 2022, 4249843.

+ Open protocol

+ Expand

Culturing Stromal and NK Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

EL08.1D2 cells stromal cells (a gift from Dr. J. Miller, University of Minnesota) were maintained on gelatinized culture flasks at 32°C in 40.5% α-MEM (Life Technologies), 50% Myelocult M5300 (STEMCELL Technologies), 7.5% heat-inactivated FCS (Atlanta Biologicals) with β-mercaptoethanol (10⁻⁵ M), Glutamax (Life Technologies, 2 mM), penicillin/streptomycin (Life Technologies, 100 U/ml), and hydrocortisone (Sigma, 10⁻⁶ M). Culture media was supplemented with 20% conditioned supernatant from previous EL08.1D2 cultures.
NK92 cells (ATCC) were maintained in 10% FBS Myelocult H5100 (Stemcell Technologies) with IL-2 (200 U/ml). Cell lines were authenticated by flow cytometry and confirmed monthly to be mycoplasma free.

Lee B.J., Solloa E.H., Shannon M.J, & Mace E.M. (2020). Generation of cell-derived matrices that support human NK cell migration and differentiation. Journal of leukocyte biology, 108(4), 1369-1378.

+ Open protocol

+ Expand

Long-Term Culture Assay of Meis-Deficient Hematopoietic Stem Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

S17 stromal line cells [12 (link)] were irradiated at 2000cGy and seeded at 1.5x10⁴ cell per well into a flat-bottom tissue culture treated 96-well plate. Cultures were then seeded with un-separated BM cells at various concentrations (3x10⁴, 1.5x10⁴, 7.5x10³, 3.75x10³) from induced ERTCre/Meis^+/-, ERTCre/Meis^-/-, MxCre/Meis^+/- or MxCre/Meis^-/-mice. Cells were prepared in mouse MyeloCult™ M5300 (STEMCELL) supplemented with freshly prepared 10⁻⁶ M hydrocortisone (21-hemisuccinate sodium salt)(mLTCM). Weekly one-half media changes were performed according to manufacturer’s protocol. Following 4 weeks in culture, adherent and non-adherent cells were harvested from individual wells and plated in methycellulose media supplemented with cytokines (CAT:3434, STEMCELL Technologies), then cultured as described for CFC assays. Colony numbers were counted and the wells were recorded as negative if no colony was present and positive if ≥ 1 colony was present. LTC-IC frequency and test for significant differences between groups was performed using the Extreme Limiting Dilution Analysis (ELDA) online tool (http://bioinf.wehi.edu.au/software/elda) [11 (link)].

Miller M.E., Rosten P., Lemieux M.E., Lai C, & Humphries R.K. (2016). Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion. PLoS ONE, 11(3), e0151584.

+ Open protocol

+ Expand

Embryonic Hematopoietic Stem Cell Isolation

Check if the same lab product or an alternative is used in the 5 most similar protocols

AGM explant (AGM^ex) culture was performed as previously described^{2 (link)}. In brief, AGMs were deposited on a nylon membrane (Millipore) placed on metallic supports and cultured in MyeloCult M5300 or H5100 (Stem Cell Technologies) supplemented with 10 µM hydrocortisone (Sigma-Aldrich). After 6 or 12 hours or 1-4 days culture, explants were collected and dissociated into single cells by 0.125% collagenase digestion (Sigma-Aldrich) for flow cytometry analysis and further culture. In some conditions, 3-methyladenine (3-MA, Merck, 1 mM), chloroquine (CQ, Selleck, 2.5 or 5 µM), bafilomycin A1 (Baf, Selleck, 50 or 100 nM), and AS1411 (the aptamer of nucleolin, 5’-GGTGGT GGTGGTTGTGGTGGTGGTGG-3’, 10 µM, from Ruibiotech) were added into the explant cultures.
Endothelial cells, pre-HSC I and pre-HSC II cells were sorted from AGM region and were co-cultured with OP9-DL1 cells (stem cell factor, 100 ng/mL; IL-3, 100 ng/mL; and Flt3-ligand, 100 ng/mL; PeproTech) or OP9 cells (stem cell factor, 20 ng/mL; IL-7, 10 ng/mL; and Flt3-ligand, 10 ng/mL; PeproTech) for 7 days as previous report^{62 (link)} and cells were harvested by mechanical pipetting for flow cytometric analysis.

Liu Y., Shi L., Chen Y., Luo S., Chen Y., Chen H., Lan W., Lu X., Cao Z., Ye Z., Li J., Yu B., Dzierzak E, & Li Z. (2024). Autophagy regulates the maturation of hematopoietic precursors in the embryo. Nature Communications, 15, 2255.

+ Open protocol

+ Expand

Short-term homing of HSCs

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

For short-term homing assay, freshly isolated HSCs were treated with 10 μM of TAT, TAT-TN13 (synthesized by Peptron, Korea) or SB203580 (S1076, Selleckchem) in HSC media (Myelocult M5300 (Stemcell Technologies) containing mSCF (250-03, PeproTech Korea) 100 ng ml⁻¹, mFlt3L (250-31 L, PeproTech Korea) 100 ng ml⁻¹ and mTPO (315-14, PeproTech Korea) 20 ng ml⁻¹) for 16 h at 37 °C, 5% CO₂. 10,000 CD45.2⁺ HSCs were i.v. injected into lethally irradiated (9 Gy) CD45.1⁺ congenic recipients. After 18 h, recipients were killed and BM cells were stained with anti-CD45.1 and CD45.2 antibodies for 25 min at 4 °C. CD45.2⁺ cells were analysed by flow cytometry and relative frequency of CD45.2⁺ cells were determined in total BM cells4 (link).

Jung H., Kim D.O., Byun J.E., Kim W.S., Kim M.J., Song H.Y., Kim Y.K., Kang D.K., Park Y.J., Kim T.D., Yoon S.R., Lee H.G., Choi E.J., Min S.H, & Choi I. (2016). Thioredoxin-interacting protein regulates haematopoietic stem cell ageing and rejuvenation by inhibiting p38 kinase activity. Nature Communications, 7, 13674.

+ Open protocol

+ Expand

Quantifying Mouse and Human LTC-IC Frequencies

Check if the same lab product or an alternative is used in the 5 most similar protocols

To assess LTC-IC frequencies, serial dilutions of FACS-sorted mouse Lin−Sca-1+c-Kit+ (LSK) cells or CD34⁺ enriched human HSPCs were plated in MyeloCult M5300 for mouse or MyeloCult H5100 for human (STEMCELL Technologies) mixed at 50:50 ratio with primary mouse stroma cell–conditioned medium or with HS5-conditioned medium for human⁷². For each cell dose (1:10, 1:20, 1:40, 1:80, 1:160 for mouse LSK cells, 1:50, 1:100, 1:200, 1:400, 1:800 for human HSPC), 10 technical replicates were performed. After 4 weeks (mouse) or 5 weeks (human) in culture with half change of the medium every week, cells in each well were used for the colony formation assay in MethoCult M3434 and MethoCult H4434 (STEMCELL technologies) for mouse and human, respectively. For CFU-C counting, in the case of mouse samples, wells containing any type of colonies were scored as positive, while in the case of human samples, only wells with GEMM colonies were scored as positive. Where indicated, the CMA activator (10μM) or the same volume of DMSO were added to the mouse LSK cells every 24 hours during the 4 weeks in culture. Supplementation of the γ-linolenic fatty acid (GLA) was done by adding 100μM GLA (Sigma, L2378) or the same volume of IMDM medium to the human HSPC cells every week for 5 weeks.

Dong S., Wang Q., Kao Y., Diaz A., Tasset I., Kaushik S., Thiruthuvanathan V., Zintiridou A., Nieves E., Dzieciatkowska M., Reisz J., Gavathiotis E., D’Alessandro A., Will B, & Cuervo A. (2021). Chaperone-mediated autophagy sustains hematopoietic stem cell function. Nature, 591(7848), 117-123.

+ Open protocol

+ Expand

Bile Acid Effects on Bone Marrow Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Irradiated AFT024 cells were seeded to confluency in 24-well plates in Dulbecco modified Eagle medium + 10% FBS + 0.05 mM 2-mercaptoethanol in a 33°C, 5% CO₂ incubator. Complete media was made adding 0.22 μm filter–sterilized minimum essential medium α with nucleosides (StemCell Technologies) containing 1mM hydrocortisone (StemCell Technologies) to Myelocult M5300 (StemCell Technologies) for a final concentration of 1 μM hydrocortisone. AFT024 media was removed from the wells, and 200 000 whole bone marrow cells in 1 mL of complete media were added to each well. Cells were cultured for 1 week either in control media or in the presence of DCA (35 μM) or LCA (18 μM). After 1 week, media were removed and placed into a collection tube, adherent cells were washed with Hanks balanced salt solution (StemCell Technologies). Hanks balanced salt solution was removed and placed into the collection tube, and cells were treated with Trypsin-EDTA (0.05%; Thermo Fisher Scientific). Trypsinization was halted by washing cells with heat-inactivated FBS, followed by IMDM + 2% FBS. This was placed back into the collection tube and centrifuged to form pellet cells. The media were decanted via aspiration. Cells were resuspended in FACS buffer (PBS + 1% FBS) and stained for spectral flow cytometry.

Thompson B., Lu S., Revilla J., Uddin M.J., Oakland D.N., Brovero S., Keles S., Bresnick E.H., Petri WA J.r, & Burgess S.L. (2023). Secondary bile acids function through the vitamin D receptor in myeloid progenitors to promote myelopoiesis. Blood Advances, 7(17), 4970-4982.

+ Open protocol

+ Expand

Stromal Cell Culture Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

The UG26‐1B6 and KH23 stromal cell lines were grown at 33°C in medium containing 50% Myelocult M5300 (Stem Cell Technologies), 35% α‐MEM (Invitrogen), 15% fetal calf serum (Sigma Aldrich), 0.5% penicillin–streptomycin (Sigma Aldrich), and 10 μM β‐mercaptoethanol.

Fitch S.R., Kapeni C., Tsitsopoulou A., Wilson N.K., Göttgens B., de Bruijn M.F, & Ottersbach K. (2019). Gata3 targets Runx1 in the embryonic haematopoietic stem cell niche. Iubmb Life, 72(1), 45-52.

+ Open protocol

+ Expand

Coculture Assay for Hematopoietic Progenitors

Check if the same lab product or an alternative is used in the 5 most similar protocols

For coculture assays, mMSCs transduced with each lentiviral vector were irradiated with 15 Gy 1 day before coculture. Murine hematopoietic progenitors were cocultured with mMSCs for 5 days in MyeloCult M5300 (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with antibiotic-antimycotic (Gibco), 100 ng/ml human Flt-3 ligand (FL, ProSpec-Tany TechnoGene, Ltd., Rehovort, Israel), 100 ng/ml mouse Stem Cell Factor (mSCF, ProSpec), 50 ng/ml human thrombopoietin (hTPO, ProSpec) and 10⁻⁶ M hydrocortisone (HC, STEMCELL Technologies). Wnt3a (10 ng/ml or 100 ng/ml, R&D Systems, Minneapolis, MN) or Wnt5a (10 ng/ml, R&D Systems) was added to the medium for coculturing, and BSA (Sigma Aldrich) was used to treat the control group. For colony forming assays of hematopoietic progenitors, hematopoietic cells were plated in methylcellulose media (MethoCult, STEMCELL Technologies) containing cytokines for 14 days and analyzed for colony numbers.

Jeong S.Y., Lyu J., Kim J.A, & Oh I.H. (2020). Ryk modulates the niche activity of mesenchymal stromal cells by fine-tuning canonical Wnt signaling. Experimental & Molecular Medicine, 52(7), 1140-1151.

+ Open protocol

+ Expand

Long-term Hematopoietic Stem Cell Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

LTC-IC assay was performed to determine the presence of long-term HSCs. For this purpose, 5×10³ CD34⁺ cells at the day of isolation and after 10 days of treatment were cultured in Myelocult M5300 (Stem Cell Technologies, Canada) on an irradiated stromal cell feeder and incubated at 37˚C and 5% CO₂. The medium was changed every four days. After six weeks of culture, the cells were suspended with trypsin and 2×10³ cells were cultured in Methocult for the CFU assay. Colonies were counted after 14 days of culture as long-term HSCs by invert microscope.

Oubari F., Amirizade N., Mohammadpour H., Nakhlestani M, & Zarif M.N. (2015). The Important Role of FLT3-L in Ex Vivo Expansion of Hematopoietic Stem Cells following Co-Culture with Mesenchymal Stem Cells. Cell Journal (Yakhteh), 17(2), 201-210.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!