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Megacult c medium

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Megacult-C medium is a specialized cell culture medium designed for the expansion and maintenance of hematopoietic stem and progenitor cells. It is optimized to support the proliferation and differentiation of these cell types.

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Lab products found in correlation

Methocult medium, by STEMCELL (3 mentions) Rhil 6, by R&D Systems (1 mentions) Mil 3, by R&D Systems (1 mentions) Mil 3, by STEMCELL (1 mentions) Rhepo, by Johnson & Johnson (1 mentions) Inverted infinity, by Thermo Fisher Scientific (1 mentions) Phase contrast microscope, by Thermo Fisher Scientific (1 mentions) Random primer, by Thermo Fisher Scientific (1 mentions) Trizol rna isolation reagent, by Thermo Fisher Scientific (1 mentions) Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)

9 protocols using megacult c medium

1

Megakaryocyte Progenitor Assay Protocol

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For mouse CFU-Mk, 1.2 × 106 cultured c-Kit+ cells were collected and cultured in 1.7 ml MegaCult-C medium (STEMCELL Technologies, #04900) supplemented with cytokines (50 ng/ml hTPO + 20 ng/ml hIL-6 + 10 ng/ml mIL-3) in IMDM and collagen solution to assess the colony formation of Mk progenitors (3 × 105 cells for each chamber), according to the manufacturer’s instructions. Colonies were scored after 6–8 days with an inverted microscope (Nikon Ti-U).
For human CFU-Mk, 2 × 105 cultured UCB CD34+ cells were collected and cultured in a 2 ml MegaCult-C medium with cytokines (STEMCELL Technologies, #04901) supplemented with collagen solution to assess the colony formation of Mk progenitors (5 × 104 cells for each chamber), according to the manufacturer’s instructions. Colonies were scored after 10–12 days with an inverted microscope (Nikon Ti-U). Microscopically magnified colonies images were collected by NIS-Elements F Software (v4.00.06).
Li Y., He M., Zhang W., Liu W., Xu H., Yang M., Zhang H., Liang H., Li W., Wu Z., Fu W., Xu S., Liu X., Fan S., Zhou L., Wang C., Zhang L., Li Y., Gu J., Yin J., Zhang Y., Xia Y., Mao X., Cheng T., Shi J., Du Y, & Gao Y. (2023). Expansion of human megakaryocyte-biased hematopoietic stem cells by biomimetic Microniche. Nature Communications, 14, 2207.
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2

Assaying Hematopoietic Progenitor Cells

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Assays of colony forming unit megakaryocyte (CFU-MK) and colony forming unit myeloid (CFU-myeloid) cells were performed on mouse lineage negative cells. For CFU-MK assay, 5,000 bone marrow lineage negative cells were seeded in Megacult-C medium (Stemcell Technologies) supplemented with 10ng/ml mouse IL-3, 10 ng/ml human IL-6, and 50 ng/ml of mouse THPO and cultured in the presence of DMSO or chemical inhibitors for 7–9 days. For CFU-myeloid assay, 2,000 bone marrow lineage negative cells were seeded in MethoCult medium (Stemcell Technologies) supplemented with 10ng/ml mouse IL-3, 10 ng/ml human IL-6, 10 ng/ml mouse SCF and 10 ng/ml of mouse granulocyte-monocyte colony stimulating factor (GM-CSF), and cultured in the presence of DMSO or chemical inhibitors for 7 days. Slides of Megacult cultures were fixed with methanol and then were stained with substrates of acetylcholinesterase according to the manufacturer’s instructions. Only stained colonies were counted. For the CFU-myeloid assay, CFU-G, CFU-M and CFU-GM were enumerated on day 7 according to the manufacturer’s instructions. The number of CFU-myeloid colonies was taken as the sum of CFU-G, CFU-M and CFU-GM.
Wen Q.J., Yang Q., Goldenson B., Malinge S., Lasho T., Schneider R.K., Breyfogle L.J., Schultz R., Gilles L., Koppikar P., Abdel-Wahab O., Pardanani A., Stein B., Gurbuxani S., Mullally A., Levine R., Tefferi A, & Crispino J.D. (2015). Targeting megakaryocytic induced fibrosis by AURKA inhibition in the myeloproliferative neoplasms. Nature medicine, 21(12), 1473-1480.
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3

Assaying Hematopoietic Progenitor Cells

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Assays of colony forming unit megakaryocyte (CFU-MK) and colony forming unit myeloid (CFU-myeloid) cells were performed on mouse lineage negative cells. For CFU-MK assay, 5,000 bone marrow lineage negative cells were seeded in Megacult-C medium (Stemcell Technologies) supplemented with 10ng/ml mouse IL-3, 10 ng/ml human IL-6, and 50 ng/ml of mouse THPO and cultured in the presence of DMSO or chemical inhibitors for 7–9 days. For CFU-myeloid assay, 2,000 bone marrow lineage negative cells were seeded in MethoCult medium (Stemcell Technologies) supplemented with 10ng/ml mouse IL-3, 10 ng/ml human IL-6, 10 ng/ml mouse SCF and 10 ng/ml of mouse granulocyte-monocyte colony stimulating factor (GM-CSF), and cultured in the presence of DMSO or chemical inhibitors for 7 days. Slides of Megacult cultures were fixed with methanol and then were stained with substrates of acetylcholinesterase according to the manufacturer’s instructions. Only stained colonies were counted. For the CFU-myeloid assay, CFU-G, CFU-M and CFU-GM were enumerated on day 7 according to the manufacturer’s instructions. The number of CFU-myeloid colonies was taken as the sum of CFU-G, CFU-M and CFU-GM.
Wen Q.J., Yang Q., Goldenson B., Malinge S., Lasho T., Schneider R.K., Breyfogle L.J., Schultz R., Gilles L., Koppikar P., Abdel-Wahab O., Pardanani A., Stein B., Gurbuxani S., Mullally A., Levine R., Tefferi A, & Crispino J.D. (2015). Targeting megakaryocytic induced fibrosis by AURKA inhibition in the myeloproliferative neoplasms. Nature medicine, 21(12), 1473-1480.
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4

Long-Term Culture of Sorted Megakaryocytes

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Sorted MEPs were thawed and counted using a hemocytometer. For each sample, 50 sorted MEPs were resuspended in 30 µL of MegaCultC medium (Stem Cell Technologies) supplemented with cytokines and mixed with collagen as described above and detailed in the text. A total volume of 15 µL was plated in the center of a MatTek dish (containing a coverslip in place of a plastic bottom for better imaging resolution) and sandwiched under a sterile 12-mm round coverslip to create minimal focal range in the z-axis. Samples were incubated for 45 min to allow for collagen polymerization. An additional 1.5 mL of MegaCultC medium, cytokines, and collagen were added on top of the second coverslip to prevent evaporation and support a long-term culture. Samples were incubated without light exposure at 5% CO2 and 37 °C for 24 h before beginning imaging.
Scanlon V.M., Thompson E.N., Lawton B.R., Kochugaeva M., Ta K., Mayday M.Y., Xavier-Ferrucio J., Kang E., Eskow N.M., Lu Y.C., Kwon N., Laumas A., Cenci M., Lawrence K., Barden K., Larsuel S.T., Reed F.E., Peña-Carmona G., Ubbelohde A., Lee J.P., Boobalan S., Oppong Y., Anderson R., Maynard C., Sahirul K., Lajeune C., Ivathraya V., Addy T., Sanchez P., Holbrook C., Van Ho A.T., Duncan J.S., Blau H.M., Levchenko A, & Krause D.S. (2022). Multiparameter analysis of timelapse imaging reveals kinetics of megakaryocytic erythroid progenitor clonal expansion and differentiation. Scientific Reports, 12, 16218.
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5

Cell Growth in Methocult and Megacult

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Harvested cells were grown in Methocult Enriched medium (H4435) and Megacult-C medium (04973, Stem Cell Technologies) as per manufacturer's instructions. Refer to Supplemental Information for more details.
Tursky M.L., Loi T.H., Artuz C.M., Alateeq S., Wolvetang E.J., Tao H., Ma D.D, & Molloy T.J. (2020). Direct Comparison of Four Hematopoietic Differentiation Methods from Human Induced Pluripotent Stem Cells. Stem Cell Reports, 15(3), 735-748.
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6

Hematopoietic Progenitor Colony Assay

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CFU-GEMM, CFU-GM and BFU-E colony formation was assessed in vitro using washed suspensions of 5 X104 marrow cells or 3 X 105 spleen cells suspended in IMDM and plated in 1% methylcellulose with 30% FBS (Methocult medium, catalogue #3534, STEMCELL Technologies, Vancouver, BC Canada) containing 10 ng/μL of mIL3 (catalogue #02733, STEMCELL), 10 ng/μL of rhIL6 (#206-IL-010, R&D SYSTEMS, Minneapolis, MNUSA), 50ng/μL of THPO (#288-TP-005, R&D) and 3 U/μL of rhEPO (Johnson and Johnson, New Brunswick, NJ USA). All colony-forming assays were performed between weeks 15–18. Colony-forming assays were performed in triplicate with at least 3 replicate experiments and colony formation was assessed at 7 days. CFU-Mk colony formation was assessed using 5 X104 washed marrow cells or 3 X 105 spleen cells suspended in IMDM and plated in methylcellulose (Megacult-C medium, # 04850, STEMCELL) containing 10 ng/μL of mIL3, 10 ng/μL of rhIL6 and 50ng/μL of THPO, R&D). Colony number was assessed at 7 days after staining with acetylcholinesterase activity as described in the Megacult-C protocol for murine cells.
Spivak J.L., Merchant A., Williams D.M., Rogers O., Zhao W., Duffield A., Resar L.S., Moliterno A.R, & Zhao Z.J. (2020). Thrombopoietin is required for full phenotype expression in a JAK2V617F transgenic mouse model of polycythemia vera. PLoS ONE, 15(6), e0232801.
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7

Evaluating Hematopoietic Progenitor Colonies

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Human HSCs (Lin−CD34+CD38−CD49f+) and MPPs (Lin−CD34+CD38−CD49f−) were FACS-sorted and cultured in rhTPO-free Megacult-C medium (Stem Cell Technologies) to assess colony formation of megakaryocyte progenitors, according to manufacturer’s instructions. Colonies were scored after 10–12 days using Inverted Infinity and Phase Contrast Microscope (Fisher Scientific). To assess the colony formation of myeloid and erythroid progenitors, stem and progenitor cells were plated in HSC003 (human) or HSC007 (mouse) methylcellulose medium according to the manufacturer’s recommendation (R&D Systems). Colonies were scored after 14–16 days for human and 8–12 days for mouse using Inverted Infinity and Phase Contrast Microscope (Fisher Scientific). In the serial replating assay, methylcellulose medium from primary plating was dissolved in PBS to dissociate the colonies into a single cell suspension. Subsequently, 20,000 cells from the previous plating were serially replated in 1 mL of HSC003 or HSC007 medium for human and mouse cells, respectively.
Kao Y.R., Chen J., Narayanagari S.R., Todorova T.I., Aivalioti M.M., Ferreira M., Ramos-Marques P., Pallaud C., Mantzaris I., Shastri A., Bussel J.B., Verma A., Steidl U, & Will B. (2018). Thrombopoietin receptor-independent stimulation of hematopoietic stem cells by eltrombopag. Science translational medicine, 10(458), eaas9563.
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8

Megakaryocyte Colony-Forming Assays and Gene Expression Analysis

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CFU-MKs assays were performed with fresh bone marrow cells using MegaCult-C medium (Stem Cell Technologies) supplemented with 50 ng/ ml rhTPO, 20 ng/ml rhIL-6, 10 ng/ ml rmIL-3, and 50 ng/ ml rhIL-11 according to the manufacturer's instruction. After 8 days of incubation, cultures were fixed, and megakaryocyte colonies were stained for acetylcholinesterase activity. Colonies containing more than three megakaryocytes were scored as CFU-MKs.
Quantitative (q)RT-PCR and Immunoblot assay Total RNA was isolated with TRIzol RNA Isolation Reagents (Thermo Fisher) and treated with DNaseI. Reverse transcription was performed with Superscript II Reverse Transcriptase with random primers (Thermo Fisher). qRT-PCR was performed using SYBR Green PCR master mix on an ABI Prism 7000 with appropriate primers. The primers of Hira mRNA detection are: mHiraExon7-F: 5 0 -AA CTCTGAGAGGTCATTCTG-3 0 ; mHiraExon7-R: 5 0 -CTTGGTGATGCTAGTCTCTA-3 0 ; mHiraExon9-F: 5 0 -TCCGGCTTAGTTGGTCA CCTG-3 0 ; mHiraExon9-R: 5 0 -CACAACAGTCACAGCTTTCC-3 0 . The primers of other genes are purchased from Sigma. Relative expression values were calculated by the 66CT method, and normalized by Hprt. Immunoblot assays were performed using 20 ug of nuclear extracts as in (Chen et al., 2012) . The primary antibodies used were mouse mAb Hira clone WC115 (Hall et al., 2001) and TF2B (Abcam).
Chen C., Sun M.A., Warzecha C., Bachu M., Dey A., Wu T., Adams P.D., Macfarlan T., Love P, & Ozato K. (2020). HIRA, a DiGeorge Syndrome Candidate Gene, Confers Proper Chromatin Accessibility on HSCs and Supports All Stages of Hematopoiesis. Cell reports, 30(7).
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9

In vitro Colony-Forming Unit-Megakaryocyte Assay

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In vitro Culture of Colony-Forming Unit- Megakaryocyte Assay (CFU-Mk) assay was done using MegaCult-C medium (Stem Cell Technologies) according to its technical manual (version 3.3.0). The cytokines used were TPO (50 ng ml−1), IL-3 (10 ng ml−1), IL-6 (20 ng ml−1) and IL-11 (50 ng ml−1). 50,000 cells (of total supernatant cells or sorted cells) were cultured in the MegaCult medium containing the cocktail of growth factors mentioned above in duplicates. Doxycycline (dox, 4 μg ml−1) were applied at the beginning of CFU-MK assay for inducible factors. By day 12–16, the CFU-Mks/CFU-mixs colonies were fixed in acetone, stained with AchE and recorded according to protocol from kit.
Cheng H., Ang H.Y., A. EL Farran C., Li P., Fang H.T., Liu T.M., Kong S.L., Chin M.L., Ling W.Y., Lim E.K., Li H., Huber T., Loh K.M., Loh Y.H, & Lim B. (2016). Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors. Nature Communications, 7, 13396.
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