DNA fragmentation was analyzed using the NucleoCounter® NC-3000™ (Chemometec, Allerod, Denmark) according to manufacturer’s protocol with minor adjustments. Matrigel was digested using TrypLE™ Express Enzyme (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min at 37 °C. Organoids were manually disrupted and washed using PBS. After at least 12 h of fixation in 70% Ethanol, cells were stained in DAPI (4′,6-Diamidin-2-phenylindol) staining solution (Solution 3, #910-3003, Chemometec, Allerod, Denmark) and finally analyzed using the NC-Slide A8™ (Chemometec, Allerod, Denmark).
Solution 3
Manufactured by ChemoMetec
Sourced in Denmark
Solution 3 is a laboratory instrument designed for automated cell analysis and counting. It utilizes advanced technology to provide accurate and reliable measurements of various cell parameters.
Automatically generated - may contain errors
Lab products found in correlation
Nc 3000, by ChemoMetec (3 mentions)
Nucleocounter nc 3000, by ChemoMetec (2 mentions)
Via1 cassette, by ChemoMetec (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Tryple express enzyme, by Thermo Fisher Scientific (1 mentions)
Nc slide a8, by ChemoMetec (1 mentions)
Nucleoview nc 3000, by ChemoMetec (1 mentions)
Halt phosphatase inhibitor single use cocktail, by Thermo Fisher Scientific (1 mentions)
Nc slides a8, by ChemoMetec (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
Penicillin g, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Amphotericin b solution, by Merck Group (1 mentions)
Nc slides a2 and a8, by ChemoMetec (1 mentions)
Solution 8, by ChemoMetec (1 mentions)
L 3 4 dihydroxyphenylalanine l dopa, by Merck Group (1 mentions)
Solution 5 vb 48 pi ao, by ChemoMetec (1 mentions)
9 protocols using solution 3
1
Quantification of Organoid DNA Fragmentation
2
Comprehensive Cell Health Assessment using NC-3000
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Nucleocounter NC-3000 (ChemoMetec, Lillerød, Denmark) controlled by the NucleoView NC-3000 Software was applied to assess cell proliferation rate, cell membrane damage and DNA fragmentation.
Cell count and viability assay is based on the staining of non-fixed cells with acridine orange (AO) and DAPI. AO is a membrane-permeable dye that binds to nucleic acids, staining all the cells in a population. DAPI cannot penetrate the cell membrane, hence it only stains cells with a damaged cell membrane (dead cells). After the treatment, cells were trypsynized, centrifuged and resuspended in the culture medium. Next, cell suspension was loaded into cassette containing the stains and immediately analyzed by the NC-3000 system.
DNA fragmentation was evaluated following a previously described method [35 (link)]. In brief, following the treatment, cells were fixed with ice-cold 70% ethanol at 4 °C for 24 h, stained with Solution 3 (ChemoMetec, Lillerød, Denmark) for 5 min at 37 °C, and then analyzed using the fluorescent cytometer.
Cell count and viability assay is based on the staining of non-fixed cells with acridine orange (AO) and DAPI. AO is a membrane-permeable dye that binds to nucleic acids, staining all the cells in a population. DAPI cannot penetrate the cell membrane, hence it only stains cells with a damaged cell membrane (dead cells). After the treatment, cells were trypsynized, centrifuged and resuspended in the culture medium. Next, cell suspension was loaded into cassette containing the stains and immediately analyzed by the NC-3000 system.
DNA fragmentation was evaluated following a previously described method [35 (link)]. In brief, following the treatment, cells were fixed with ice-cold 70% ethanol at 4 °C for 24 h, stained with Solution 3 (ChemoMetec, Lillerød, Denmark) for 5 min at 37 °C, and then analyzed using the fluorescent cytometer.
3
Melanoma Cell DNA Fragmentation Analysis
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The DNA fragmentation analysis of melanoma cells was performed using the fluorescence image cytometer NucleoCounter® NC-3000™. After the treatment, the cells were detached, counted and fixed with 70% cold ethanol (24 h at 0–4 °C). Afterwards, the samples were washed with PBS, centrifuged and stained with Solution 3 (ChemoMetec, Lillerød, Denmark) containing DAPI and 0.1% triton X-100 in PBS. The analysis was made according to the DNA Fragmentation Assay protocol.
4
Cell Cycle Analysis by Flow Cytometry
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Cell cycle phase distribution was analyzed by the use of fluorescence image cytometer NucleoCounter NC-3000. The analysis is based on differences in DNA content between the pre-replicative phase (G1/G0) cells versus the cells that actually replicate DNA (S phase) versus the post-replicative plus mitotic (G2/M) cells [25 (link)]. In brief, cells were trypsinized, counted, and fixed with ice-cold 70% ethanol. After washing, cell pellets were stained with Solution 3 (ChemoMetec, Lillerød, Denmark) containing DAPI and Triton X-100, loaded into NC-Slides (ChemoMetec), and analyzed using the NC-3000 system where cellular fluorescence was quantified into DNA content histograms. Markers in the displayed histograms were used to identify cells in different cell cycle stages or to demarcate apoptotic cells with fragmented DNA having less than 1 DNA equivalent (sub-G1 cells).
5
Cell Fixation and Analysis via Image Cytometry
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The cells were trypsinized and centrifuged (5 min, 500× g) followed by washing twice with phosphate-buffered saline (PBS). The suspended pellet (500 µL PBS) was fixed in 70% ethanol (4.5 mL) and stored (4 °C) until the day of analysis. After centrifugation (5 min, 500× g), ethanol-fixed cells were mixed with Solution 3 (ChemoMetec, Allerod, Denmark), incubated (37 °C, 5 min), and analyzed with an image cytometer (NC-3000, ChemoMetec, Allerod, Denmark).
6
Cell Cycle Analysis of PRP-Treated HaCaT Cells
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HaCaT cells were PRP-treated (0.5–5%) for 24 h. After incubation, cells were trypsinized, centrifuged (5 min, 500× g), and washed twice with PBS. The pellet was suspended in 0.5 mL of PBS, fixed in 4.5 mL of ice-cold 70% ethanol, and stored at 4 °C until analysis. On the day of analysis, ethanol-fixed cells were centrifuged (5 min, 500× g), washed in PBS, and then suspended in Solution 3 (ChemoMetec, Allerod, Denmark) followed by incubation at 37 °C for 5 min. The fixed cell cycle analysis was conducted using an image cytometer (NC-3000, ChemoMetec, Allerod, Denmark).
7
Comprehensive Protein Extraction and Quantification
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Dulbecco’s phosphate-buffered saline, Halt Protease Inhibitor Cocktail, Halt Phosphatase Inhibitor Single-Use Cocktail, Pierce BCA Protein Assay Kit, and Trypsin/EDTA solution were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Amphotericin B, penicillin G, and RIPA Buffer were purchased from Sigma-Aldrich Inc. (St. Luis, MO, USA). NC-Slides A8, Solution 3, and Via1-Cassettes were obtained from ChemoMetec (Lillerød, Denmark). Growth media (DMEM and RPMI 1640) and fetal bovine serum were acquired from CytoGen (Zgierz, Poland). Neomycin sulfate was obtained from 1Amara (Kraków, Poland). The remaining chemicals were purchased from POCH S.A. (Gliwice, Poland).
8
Cell Cycle Analysis of MCF-7 Cells
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Analysis of the cell cycle of MCF-7 and MCF-7shPRODH/POX cells was performed as follows. Non-treated and Cx-treated (5–25 µM, 24 h) cells were trypsinized and centrifuged (5 min, 500× g) followed by washing twice with PBS. The suspended pellet (500 µL PBS) was fixed in 70% ethanol (4.5 mL) and stored (4 °C) until the day of analysis. After centrifugation (5 min, 500× g), ethanol-fixed cells were mixed with Solution 3 (ChemoMetec, Allerod, Denmark), incubated (37 °C, 5 min), and analyzed with an image cytometer (NC-3000, ChemoMetec, Allerod, Denmark).
9
Meloxicam Effects on Fibroblast and Melanocyte
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Meloxicam was obtained from Boehringer Ingelheim (Budapest, Hungary). Fibroblasts Growth Medium, amphotericin B solution (250 µg/mL), penicillin, phosphate-buffered saline (PBS) and L-3,4-dihydroxyphenylalanine (L-DOPA) were purchased from Sigma Aldrich Inc. (St. Louis, MO, USA). Neomycin sulfate was acquired from Amara (Kraków, Poland). An M-254 growth medium and a human melanocyte growth supplement-2 (HMGS-2) were obtained from Cascade Biologics (Portland, OR, USA). Trypsin/EDTA was obtained from Cytogen (Zgierz, Poland). Via-1-Cassettes™ (acridine orange and DAPI fluorophores), NC-slides A2 and A8, as well as Solution 3 (1 µg/mL DAPI, 0.1% triton X-100 in PBS), Solution 5 (VB-48™ PI AO), Solution 7 (200 µg/mL JC-1) and Solution 8 (1 µg/mL DAPI in PBS) were purchased from ChemoMetec (Lillerød, Denmark). H2DCFDA reagent was acquired from Thermo Fisher Scientific Inc. (Waltham, MA, USA). WST-1 cell proliferation reagent was obtained from Roche GmbH (Mannheim, Germany). Other chemicals were obtained from POCH S.A. (Gliwice, Poland).
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