Cord blood was obtained from the Clinic of Gynecology of the Medical University of Vienna. All donors gave their written informed consent. CD34+ hematopoietic stem cells were isolated from cord blood with CD34 MACS magnetic beads (Miltenyi, Bergisch Gladbach, Germany). CD34+ cells were cultured with 50 ng mL−1 thrombopoietin, 1 ng mL−1 stem cell factor and interleukin‐3 (Miltenyi) in Stem Pro 34 medium (Invitrogen, Carlsbad, CA, USA) at 37 °C in 5% CO2 for 12 days to obtain mature culture differentiated megakaryocytes. Mature megakaryocytes were stained with CD41, CD14 and CD45 antibodies (eBioscience, San Diego, CA, USA), and their purity was analyzed by fluorescence‐activated cell sorting (FACS). Cultures containing > 3% contaminating leukocytes were used for our experiments. Cell lines CHRF, Meg‐01, HL‐60 and HepG2 were purchased from ATCC. They were grown in RPMI medium or Dulbecco's modified Eagles's medium (Invitrogen) supplemented with 10% fetal bovine serum and gentamicin (Gibco, Carlsbad, CA, USA) at 37 °C in 5% CO2.
Meg 01
Manufactured by American Type Culture Collection
Sourced in United States
The MEG-01 is a piece of laboratory equipment used for cell culture. It provides a controlled environment for the growth and maintenance of cells, including temperature and gas regulation. The MEG-01 is designed to support the cultivation of various cell types in a sterile and carefully monitored setting.
Automatically generated - may contain errors
Lab products found in correlation
Hl 60, by American Type Culture Collection (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Thp 1, by American Type Culture Collection (3 mentions)
Mv4 11, by American Type Culture Collection (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Ku812, by American Type Culture Collection (3 mentions)
Kasumi 1, by American Type Culture Collection (2 mentions)
Meg 01, by Merck Group (2 mentions)
Silencer select sirna custom synthesis product, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Glycerophosphate, by Merck Group (2 mentions)
Hek293t, by American Type Culture Collection (2 mentions)
Hepg2, by American Type Culture Collection (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Stempro 34 medium, by Thermo Fisher Scientific (1 mentions)
Stem cell factor (scf), by Miltenyi Biotec (1 mentions)
Dulbecco s modified eagles s medium, by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by Miltenyi Biotec (1 mentions)
Thrombopoietin, by Miltenyi Biotec (1 mentions)
30 protocols using meg 01
1
Megakaryocyte Differentiation from Cord Blood
2
Culturing Diverse Cell Lines
3
Analyzing Platelet Activation in Megakaryocytes
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Human megakaryocytes (Meg-01, ATCC: CRL-2021), were nucleofected using Lonza kit C with 3 µg of WT-TP or A160T per 100,000 cells following recommended manufacturers protocol and as described previously [16] (link). Briefly, nucleofected cells were incubated for 24 hours at 37°C. Then platelet like particles (PLPs) was collected from the media of the nucleofected megakaryocytes. PLPs were incubated vehicle (buffer or water alone) for 15 mins at room temperature. The PLPs were incubated in PBS containing PE-anti-CD41 to label all PLPs and FITC-anti-CD62P to label activated particles and incubated for 1 hour at 4°C. The samples were washed 2 times with PBS spun down and resuspended in PBS for Flow cytometry analysis. Similarly, to assess whether the drugs decrease CD62P activation in PLPs, 1 µM concentration of each compounds, SQ 29,548, Ramatroban, L-670596 and Diclofenac on WT-TP and A160T were tested.
4
Megakaryocytic Differentiation and SON Knockdown
5
Megakaryocyte Differentiation from Cord Blood CD34+ Cells
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Purified cord blood (CB) CD34+ hematopoietic stem cells were purchased from StemCell Technologies and cultured in StemSpan II media with the MK supplemental cytokines (StemSpan II, Stemcell Technologies, Inc.), according to the culture and differentiation protocols from Stemcell Technologies (Stemcell Technologies Inc. Cambridge, MA). These CD34+-derived MKs are referred to as SC-MKs throughout the paper. A megakaryoblastic cell line (Meg-01) was purchased from ATCC (American Type Culture Collection, Manassas, VA) and cultured in RPMI with 10% FBS, according to ATCC’s standard culture protocols. Meg-01 cells were used for experiments for both optimization of protocols prior to repetition with the SC CD34 cell line or when a high concentration of cells was needed for multiple replicates, such as the chemotaxis assays. Because Meg-01 cells are from a megakaryoblastic cell line and therefore may not exhibit the same behavior or receptors as the fully-mature, non-eternal MK cells, CD34+-derived MK cells were used as an additional MK model to further support our findings, although these cells may also not display the same behaviors as circulating MKs (48 (link), 49 (link)).
6
Cell Culture and Dengue Virus Assay
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Baby hamster kidney fibroblast cells (BHK-21) (ATCC, Manassas, VT, USA) and African green monkey kidney epithelial cells (Vero) (ATCC, Manassas, VT, USA) were cultured and maintained in Dulbecco’s modified Eagle´s medium (DMEM) (Cytiva, Chicago, IL, USA) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Gibco, Waltham, MA, USA). Philadelphia chromosome-positive chronic myelogenous leukemia bone marrow cells (Meg-01) (ATCC, Manassas, VT, USA) were cultured and maintained in RPMI-1640 medium (RPMI) (Cytiva, Chicago, IL, USA) supplemented with 10% heat-inactivated FBS. All the cells were grown at 37 °C with 5% CO2. Subculturing procedures were performed according to the guidelines provided by ATCC. Dengue virus serotype 2 (DENV, 16681 strain) was used in these experiments [59 (link)]. DENV was propagated in Vero cells, and the viruses were titrated by plaque assay and used for the following experiments.
7
Cell Culture Conditions for MEG-01 and UKE-1
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MEG-01 (ATCC) and UKE-1 (Coriell Institute) cells were cultured in RPMI 1640 medium (ATCC modification) and RPMI 1640 medium (Thermo Fisher Scientific), respectively, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All cell lines were maintained at 37°C and 5% CO2 and regularly tested for mycoplasma.
8
Dengue Virus Serotype 2 Propagation and Titration
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Dengue virus serotype 2 (DENV-2) (IND/P23085/1960 strain, Gene Bank accession no. JQ922552.1) was used in this study. The virus was cultured in C6/36 cells and concentrated through centricon-100 K before aliquoting and storing at − 80 °C. The virus was titrated by focus-forming unit (FFU) assay on Vero cells using D1-4G2-4-15 antibody13 (link). The Aedes albopictus C6/36 cells (CRL-1660, ATCC) were maintained in the L-15 medium at 28 °C The African green monkey kidney Vero cells (National Centre for Cell Science, Pune, India) were maintained in Minimal Essential Medium (MEM) (Invitrogen), and the human megakaryoblast MEG-01 cells (CRL-2021, ATCC) were maintained in RPMI 1640 medium at 37 °C with 5% CO2. All culture media were supplemented with 10% Fetal Bovine Serum (FBS, HyClone) and 1% Penicillin–Streptomycin–Glutamine.
9
Megakaryocytic Differentiation and SON Knockdown
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The cell lines, MEG-01 (non-DS AMKL), K562 (chronic myeloid leukemia; erythroblastic leukemia), Kasumi-1 (t(8;21)-positive acute myeloid leukemia) and HL60 (acute promyelocytic leukemia), were purchased from ATCC (Manassas, VA). The CMY and CMK cell lines (DS-AMKL) were kindly provided by Dr. Shai Izraeli (Tel Aviv University). The cell lines were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum and 4mM L-glutamine. For differentiation experiments, AMKL cell lines (MEG-01, CMY and CMK) were treated either with DMSO (control) or with 5 nM phorbol 12-myristate 13-acetate (PMA; Sigma Aldrich, St. Louis, MO) and incubated for 4 and 8 days to evaluate SON expression during megakaryocytic differentiation. Day 0 samples were used as a control for each treatment group. For knockdown experiments, cells were nucleofected with Amaxa Nucleofector II for siRNA transfection and incubated for 2 to 5 days depending on the purpose of experiments. The SON siRNA sequence used for nucleofection is GCAUUUGGCCCAUCUGAGAtt (Silencer Select siRNA custom synthesis product by Life Technologies/ThermoFisher, Waltham, MA) which was verified for its effectiveness and specificity in previous studies [21 (link), 28 (link)]. RUNX1 siRNA (siRNA ID s229352) and negative control siRNA (UAACGACGCGACGACGUAAtt; custom synthesis product) was purchased from ThermoFisher Scientific.
10
MEG-01 Cell Line Culture Protocol
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The human megakaryoblastic leukemia cell line MEG‐01 was obtained from the ATCC and cultured in RPMI‐1640 supplemented with 10% (vol/vol) fetal bovine serum, 1% penicillin/ streptomycin, and 0.3 μg/mL glutamine in a humidified atmosphere at 37°C and 5% CO2.2
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