A defined number of ITSCs was cultured in Afc-FEP bags 2PF-0002 (Afc) or 9.2 cm2 Tissue Culture Dishes (TPP) in standard medium supplemented with 10% BP for five days as described above. For determination of total cell numbers, counting was performed after digestion with collagenase NB4 (SERVA Electrophoresis) and harvesting at 300 × g, in a Cellometer Auto T4 device (Nexcelom Bioscience, Lawrence, MA, USA). The vitality of the cultured cells was assessed using trypan blue staining (Sigma-Aldrich) according to the manufacturer’s guidelines followed by measurement with a Cellometer Auto T4 device (Nexcelom Bioscience). Doubling time was calculated according to the formulas (doubling time = ln(2)/growth rate) and [(ell amount = cell concentration (t = 0)*egrowth rate*time). Statistical analysis of cell proliferation and vitality was performed with Graph Pad Prism (GraphPad Software, San Diego, CA, USA).
Cellometer auto t4
Manufactured by Revvity
Sourced in United States
The Cellometer Auto T4 is a compact and automated cell counter that provides accurate cell counts and viability analysis. It uses an image-based detection method to analyze cell samples and report the total cell count and viability percentage.
Automatically generated - may contain errors
Lab products found in correlation
Hrp linked anti rabbit or mouse, by Cytiva (5 mentions)
100 μm nylon mesh, by BD (4 mentions)
Trypan blue, by Merck Group (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (4 mentions)
Tryple express, by Thermo Fisher Scientific (3 mentions)
Collagenase type 4, by Merck Group (3 mentions)
Dnase 1, by Roche (3 mentions)
Chromium single cell 3 library gel bead kit v2, by 10x Genomics (3 mentions)
Chromium single cell controller, by 10x Genomics (3 mentions)
Red cell lysis buffer, by Thermo Fisher Scientific (3 mentions)
Differential quik stain kit, by Electron Microscopy Sciences (2 mentions)
Tryple express enzyme, by Thermo Fisher Scientific (2 mentions)
Isotonic 36 percoll, by GE Healthcare (2 mentions)
Ack lysis buffer, by Quality Biological (2 mentions)
Benzonase, by Thermo Fisher Scientific (2 mentions)
Nad nadh assay kit, by Promega (2 mentions)
Hyclone dulbecco s modified eagle s medium dmem high glucose, by Thermo Fisher Scientific (2 mentions)
Ciclosporin a, by Merck Group (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
152 protocols using cellometer auto t4
1
Efficient ITSC Cultivation and Characterization
2
Cell Detachment and Sample Preparation
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The cells were detached by adding 1 mL TrypLETM Express ([-] Phenol Red, Gibco by Life technologiesTM, Darmstadt, Germany) to the flasks. The cells were counted using Cellometer SD100 cell counting chambers (Nexcelom Bioscience, Lawrence, MA, USA) and CellometerTM Auto T4 (Nexcelom Bioscience), pelleted at 1000 g for 10 min and stored adequately until further usage.
For histological analyses, pellets were stored in 4% paraformaldehyde. For gene expression analyses, using either QuantiGene Plex Assay (Affymetrix, Santa Clara, CA, USA) or conventional PCR, cell pellets were stored at −80 °C.
For histological analyses, pellets were stored in 4% paraformaldehyde. For gene expression analyses, using either QuantiGene Plex Assay (Affymetrix, Santa Clara, CA, USA) or conventional PCR, cell pellets were stored at −80 °C.
3
Cell Proliferation and Apoptosis Assay
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The concentrations used for cell count analysis and apoptosis are based on the 72 h values of the BrdU assay. Concentrations were chosen, that lead to 50% inhibition (2.5 μM PDA-66 in PC-3 and 5 μM PDA-66 in LNCaP). These values were chosen to ensure that there would be some margin in both directions to calculate possible significances, even if the other tests would show higher or lower efficiencies. Based on this criterion, 5 μM PDA-66 should have been used for the CT1258 cell line. However, this concentration showed no effect after 24 h. As it was crucial to be able to detect effects early after exposure for the transcriptomic analysis, 15 μM was used for this cell line, the first concentration to show a significant effect in the BrdU assay after 24 h of exposure.
Cells were seeded in 24-well plates with a cell density of 5 × 104 cells per well in 1 ml culture medium and incubated overnight. Based on the proliferation assay, PC-3 cells were treated with 2.5 μM PDA-66 and 0.025% DMSO, LNCaP with 5 μM PDA-66 (0.05% DMSO), and CT1258 cells with 15 μM PDA-66 (0.15% DMSO). After 24 h, 48 h and 72 h, the cells were detached by TrypLE™ Express (Thermo Fisher Scientific Inc., Waltham, MA, USA) and counted via automatic cell counter (CellometerTM Auto T4, Nexcelom Bioscience LLC, Lawrence, MA, USA). The experiment was carried out in biological replicates three times.
Cells were seeded in 24-well plates with a cell density of 5 × 104 cells per well in 1 ml culture medium and incubated overnight. Based on the proliferation assay, PC-3 cells were treated with 2.5 μM PDA-66 and 0.025% DMSO, LNCaP with 5 μM PDA-66 (0.05% DMSO), and CT1258 cells with 15 μM PDA-66 (0.15% DMSO). After 24 h, 48 h and 72 h, the cells were detached by TrypLE™ Express (Thermo Fisher Scientific Inc., Waltham, MA, USA) and counted via automatic cell counter (CellometerTM Auto T4, Nexcelom Bioscience LLC, Lawrence, MA, USA). The experiment was carried out in biological replicates three times.
4
Cytotoxicity Evaluation of FX-9 in Cancer Cells
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The four carcinoma cell lines were seeded in 6-well plates (120 × 103 cells per well) in 3 mL culture medium and incubated overnight. On the next day, FX-9 incubation was started (PC-3, LNCaP and 0846 with 5.0 µM FX-9 and CT1258 with 2.5 µM). After 24 h, 48 h, and 72 h the cells were harvested and counted via automatic cell counter (CellometerTM Auto T4, Nexcelom Bioscience, Lawrence, MA, USA). For the benign cells, hTF-8 and 1801, 60 × 103 cells were seeded per well and treated with 5 µM FX-9 on the next day. Cells were counted after 72 h. For each FX-9 concentration and the controls, three wells were measured, and the mean value was used per experiment. Each independent experiment was performed three times.
5
Cell Counting and Preservation Protocol
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After washing with PBS to remove dead cells and trypsinization with TrypLETM Express (GibcoTM, Thermofisher Scientific) cell number was counted with an automated CellometerTM Auto T4 (Nexcelom Bioscience, Lawrence, MA, USA) and compared with untreated control. Cells were washed with PBS, centrifuged at 1000 rpm for 10 minutes (Biochrom) and stored at -80°C for further examinations (quantitative RT-PCR and protein analysis).
6
Cell Growth Assay with Metformin
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Growth curves were generated with/without clinically relevant metformin concentrations: 200uM. Numbers of live cells were determined on day 0, day 1, day 2, day 5, day 6 and day 7 (no day 7 for SK-Hep-1) (n=3), using Cellometer Auto T4 (Nexcelom Bioscience). Acquired numbers were averaged and plotted as previously described (Supplemental Fig. S1 )12 .
7
Cell Proliferation Assay with PDGF-BB
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Each well of a 12-well plate was seeded with 5000 cells. Cells were maintained in phenol red-free DMEM/F12 containing 10% steroid-free FBS under standard tissue culture conditions. Three days later when cells were approximately 60%–70% confluent, cells were growth-arrested for 2 days in DMEM/F12 containing 0.4% bovine serum albumin. Next, cells were placed in DMEM/F12 containing a low concentration (25 ng/ml) of PDGF-BB and then treated every day for 4 days with various treatments. Cells were then harvested and cell number quantified, in a blinded fashion, using a Nexcelom Cellometer Auto T4 cell counter (Nexcelom Bioscience, Lawrence, MA).
8
Bacterial Invasion Assay in Caco-2 Cells
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Bacterial invasion assays were performed using human intestinal epithelial cells (Caco-2) as described previously [32 (link)], with some modifications that were specifically employed for this study. Briefly, 105 Caco-2 cells/well were seeded in 24-well tissue culture plates and incubated at 37 °C overnight. Cells in one of the wells were counted using Cellometer Auto T4 (Nexcelom Bioscience, Lawrence, MA) and the Caco-2 cells were infected with SE819, transconjugant SE819::IncFIB, and wild type SE163A strains at multiplicity of infection (MOI) of 10. After incubation for 1 h at 37 °C, the cells were washed twice with PBS to remove bacteria that had not infected the Caco-2 cells and incubated with 200 μg/ml of gentamicin. After incubation for 1 h at 37 °C, the cells were washed twice with PBS and lysed with 0.1% chilled Triton X-100, followed by dilution and plating on TSA agar to obtain colony forming unit counts (CFUs) of bacteria following overnight incubation at 37 °C. Three replicates per strain were included and the experiments were repeated three times.
To determine influence of iron in invasion of bacteria in to host cells, wild-type (SE163A), recipient (SE819) and transconjugant (SE819::IncFIB) strains were grown overnight in iron-rich and iron-depleted media prior to infection of Caco-2 cells.
To determine influence of iron in invasion of bacteria in to host cells, wild-type (SE163A), recipient (SE819) and transconjugant (SE819::IncFIB) strains were grown overnight in iron-rich and iron-depleted media prior to infection of Caco-2 cells.
9
Embryonic and Perinatal Mouse Craniofacial Development
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Mice (Mus musculus) of the ICR strain were studied at embryonic/prenatal (E) days E13 and E15 and perinatal/postnatal (P) stage P0. The samples were obtained post mortem in agreement with the recent legislation in the Czech Republic, Act No. 359/2012 Sb, where according to paragraph 3, part t) “there is no specific requirement for approvals in case when the organ/tissue samples are collected post mortem” (which is the case of the presented investigation). Animal treatment at the IAPG runs under the approved protocols and corresponding supervision - certification 4546/2021-MZE – 18134.
Fresh post mortem mouse heads were fixed in 4% buffered paraformaldehyde (PFA), then dehydrated via gradient ethanol solutions, treated with xylene and embedded in paraffin. The MC3T3-E1 osteoblastic cell line was purchased from the European Collection of Authenticated Cell Culture (c.n. 99072810). Cells were counted by Cellometer Auto T4 (Nexcelom).
Fresh post mortem mouse heads were fixed in 4% buffered paraformaldehyde (PFA), then dehydrated via gradient ethanol solutions, treated with xylene and embedded in paraffin. The MC3T3-E1 osteoblastic cell line was purchased from the European Collection of Authenticated Cell Culture (c.n. 99072810). Cells were counted by Cellometer Auto T4 (Nexcelom).
10
Adept® Impact on Ovarian Cancer Cell Growth
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To examine the dose-dependent effect of Adept® solution on the cell growth of ovarian cancer cell lines, OVCAR-3 and A2780 cells were seeded at a density of 1.1 × 104 cells/cm2 and 5.6 × 103 cells/cm2, respectively, in triplicate, and cultured in regular medium for 3 days. Thereafter, cells were treated with either 1, 5, 10, 15 or 20% Adept® (equal to 0.04, 0.2, 0.4, 0.6 or 0.8% icodextrin) solution in regular medium for 3 days. Meanwhile, control cells were treated with culture medium alone for 3 days. Live cell numbers were counted by the Trypan blue exclusion assay using a Cellometer Auto T4 Image-based cell counter (Nexcelom Bioscience, Lawrence, MA, USA).
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