HPC assays were performed as described by the manufacturer (StemCell Technologies Inc., Grenoble, France). In short, 1000 BM- or 200 UCB-derived CD34+ or CD133+ cells (supplemented with CTLs or not) were cultured in 1.1 ml Methocult dispensed in 35-mm dishes (Greiner, Alphen a/d Rijn, The Netherlands) for 14 days at 37°C, 5% CO2. The number of erythroid, granulocyte and monocyte colonies was expressed as number of colonies formed per plate using an inverted light microscope. The HALO-96 human assay was used to detect human progenitor cell proliferation in mouse BM as described by the manufacturer (HemoGenix, Colorado, USA). Briefly, 1,25x105 BM WBC/well were plated in a 96-wells plate and incubated at 37°C, 5% CO2 for 6 days with HPC specific cytokine mix (HemoGenix). Intracellular ATP content served as substrate for a luciferin/luciferase reaction was measured as relative light units (RLU) (Centro LB 960 luminometer, Berthold Technologies, Vilvoorde, Belgium) and was determined exactly based on an ATP standard curve included into the assay.
35 mm dishes
Manufactured by Greiner
Sourced in United States, Austria
35-mm dishes are circular cell culture plates with a diameter of 35 millimeters. They are designed for the cultivation and observation of cells in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Centro lb 960 luminometer, by Berthold Technologies (1 mentions)
Transferin, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Lonza (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Harvard Bioscience (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
48 well mea plates, by Axion Biosystems (1 mentions)
Matrigel, by Corning (1 mentions)
4 protocols using 35 mm dishes
1
Hematopoietic Progenitor Cell Assays
2
Hematopoietic Progenitor Cell Assay
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After expansion on 2D or 3D surfaces/scaffolds, 1x104 cells per condition were plated in individual 35 mm dishes (Greiner) in 2 ml multi-lineage CFU media (0.75 ml FCS (Lonza), 0.5 ml Iscove's Modified Dulbecco's Media (IMDM; Gibco), 0.4 ml IMDM double conc. (Gibco), 0.25 ml 10% BSA (Stemcell; in IMDM), 20 μl Transferin (Sigma; 27 mg/ml in IMDM), 15 μl 0.093% 2-mercaptoethanol (Sigma; in PBS), 10 μl IL3 (Peprotech; 2.5 μg/ml), 10 μl IL6 (Peprotech; 2.5 μg/ml), 10 μl SCF (Peprotech; 2.5 μg/ml), 10 μl GM-CSF (Peprotech; 10 ng/ml), 100 U/ml penicillin and 100 U/ml streptomycin (Lonza), 6 μl/ml L-glutamine (Biochrom; 200mM), 5 μl/ml erythropoietin (Janssen-Cilag GmbH, in IMDM, stock 100 U)) and 400 μl agar (BD Bioscience; 200 mg/10 ml H2O) per dish. Colonies were scored after additional 14 DIV. The nature of CFUs: colony-forming unit-granulocyte/erythroid/megakaryocyte/monocyte (CFU-GEMM), colony-forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), colony-forming unit-erythroid (CFU-E), colony-forming unit-macrophage (CFU-M) and colony-forming unit-granulocyte (CFU-G) was scored and confirmed according the quality assurance procedure instruction of the University Hospital Jena.
3
Characterization of hiPSC-Derived Cardiomyocytes
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hiPSC-CMs (Pluricyte® CMs) were provided by Ncardia (Gosselies, Belgium) as cryopreserved samples, and stored in liquid nitrogen until thawed. Thawing and culturing were carried out according to the manufacturer’s protocols. Once thawed, Pluricyte® CMs were maintained in an incubator (37°C, 5% CO2), in Pluricyte® Cardiomyocyte Medium (Ncardia). Medium was refreshed at day 1 post-thaw and subsequently every other day. For manual patch clamp, the cells were seeded on 6-mm glass coverslips (VWR, Radnor, USA) coated with Matrigel (Corning Inc., New York, USA, Ref. 354277) and placed into 35-mm dishes (Greiner Bio-One, GmbH, Solingen, Germany), with a density of 300 000–400 000 cells/dish. For patch clamp, Pluricyte® CMs were tested 5–7 days after thawing. A single coverslip was placed in the recording chamber of an inverted microscope and used for about 1 h of recordings. For multi-electrode arrays (MEA), cells were seeded onto 48-well MEA plates (Axion Biosystems, Inc., Atlanta, USA) coated with fibronectin (50 µg/mL in PBS with Ca2+ and Mg2+, Sigma-Aldrich, Cat. No. F-1141), with a density of 30 000 cells/well. Multi-electrode array recordings were performed at day 8 post-thaw.
4
Retroviral Transduction and Apoptosis Assay
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For retrovirus preparation, HEKs (0.25 x 10 6 ) were seeded in 35 mm dishes (Greiner Bio-one, Kremsmünster, Austria). Cells were transfected the next day with plasmids containing the gene of interest (1.5 µg) and packaging vector pCL-Eco (1.5 µg for plasmids and 2 µg for shRNA) using X-tremeGENE. Retrovirus transduction was as described earlier (38) . After transduction live cells were selected in histopaque (1.083 g/ml density) by centrifugation at 1500 rpm for 20 min. Cells were washed twice in RPMI-CM and PBS and cultured in media supplemented with IL-2 (1 μg/ml) and IL-7 (2 ng/ml) for another 22-24 h and used for apoptosis assays. Knockdown or overexpression of targeted genes was assessed by Western Blotting.
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