Absolute number of cells was determined by whole-well imaging using the BD-PathwayBioimager855 (BD-Biosciences, [25] (link)). IPLab and AttoVision software packages (BD-Biosciences) were used for image background subtraction, segmentation and quantification. The method was validated for 96- and 384-well plates in which living and dead cells, insulin-positive cells and nuclei with thymidine-analogues were counted.
Attovision
Manufactured by BD
AttoVision is a high-performance microscopy system designed for advanced imaging applications. It features a modular architecture that allows for customization to meet specific research needs. The core function of AttoVision is to provide researchers with a powerful and versatile imaging platform for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Pathway 855, by BD (4 mentions)
Pathway 855 microscope, by BD (3 mentions)
μclear imaging plates, by Greiner (2 mentions)
Mouse anti brdu antibody, by BD (2 mentions)
Pathway 855 bioimager, by BD (1 mentions)
Diva software, by BD (1 mentions)
Anti brdu antibody, by BD (1 mentions)
β agarase, by New England Biolabs (1 mentions)
9 protocols using attovision
1
Automated Cell Counting and Quantification
2
Automated Single-Cell Immunofluorescence Imaging
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Immunofluorescence stainings were performed in 96-well μClear imaging plates (Greiner bio-one), documented and quantified on a single-cell level with the automated fluorescence microscope BD Pathway 855 using the software Attovision (BD Biosciences). Cells were fixed in 3.7% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) or, for DNA damage detection, in ice-cold methanol/acetone (1:1) for 15 min. PFA-fixed samples were washed two times with 0.1 M glycin in PBS and permeabilized in PBS with 0.1% NP-40 (each 5 min, RT). Blocking was performed in permeabilization buffer supplemented with 5% FBS for 1 h at room temperature (RT). Cells were incubated with primary antibodies (listed in Supplementary data) diluted in blocking buffer for 1 h at 37°C. After washing in blocking buffer, samples were incubated with fluorescent secondary antibodies in blocking buffer supplemented with DAPI (200 nM) for nuclear counterstain and incubated for 1 h at room temperature. After washing in permeabilization buffer, cells were kept in PBS, imaged and quantified.
3
Immunofluorescence Staining of Cells
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Immunofluorescence staining was performed in 96-well μClear imaging plates (Greiner Bio-One). Cells were fixed in 3.7% paraformaldehyde (PFA) in PBS or in ice-cold methanol/acetone (1:1) for 15 minutes. PFA-fixed samples were washed 2 times with 0.1 M glycine in PBS and permeabilized in PBS with 0.1% NP-40 (each 5 minutes, room temperature). Blocking was performed in permeabilization buffer supplemented with 5% fetal bovine serum for 1 hour at room temperature. Cells were incubated with primary antibodies diluted in blocking buffer for 1 hour at 37°C. After washing in blocking buffer, samples were incubated with fluorescent secondary antibodies in blocking buffer supplemented with 4′,6-diamidino-2-phenylindole (200 nM) for nuclear counterstain and incubated for 1 hour at room temperature. After washing in permeabilization buffer, cells were kept in PBS, imaged, and quantified on a single-cell level with the automated fluorescence microscope BD Pathway 855 using the software Attovision (BD Biosciences).
4
High-Content Immunofluorescence Imaging
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For all 96-well plate immunofluorescence assays (apoptosis, lipid loading and cell fusion), nine images per well were taken using the high content analyzer (HCA) BD pathway 855 (BD Biosciences) with 10x objective and further analyzed using Attovision and DIVA software (BD Biosciences).
5
BrdU Comet Assay for Newly Synthesized DNA
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The BrdU comet assay was performed to detect newly synthesized single strand DNA as previously described (42 (link)). Cells were labeled with 100 μM BrdU for 30 min. BrdU labeled cells were washed with PBS-, placed into fresh medium and incubated for an additional 30 min, 1 h or 2 h to mature replicating DNA strands. Labeled cells were collected, suspended in low-melting-point agarose and added to agarose-coated CometSlides. Slides were incubated in an alkaline lysis solution according to the manufacturer's protocol (Trevigen, 4250-050-K). Following electrophoresis, slides were neutralized by treatment with 0.4 M Tris–HCl, washed with PBS-, and immunostained. Incorporated BrdU substituted newly replicated DNA was detected with an anti-BrdU antibody (BD BioScience, 555627), followed by anti-mouse IgG Alexa 488 (Invitrogen, A-11029). DNA was counter-stained with DAPI and slides were dried according to the manufacturer's instructions (Trevigen, 4250-050-K). Digital images were acquired using a 20× objective lens on a BD pathway 855 microscope, which was controlled by AttoVision (Becton Dickinson). Images were analyzed by CometScore software (TriTeK).
6
Molecular Combing of DNA Replication Tracts
7
BrdU Comet Assay for DNA Damage
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The BrdU comet assay was performed as previously described 43 (link). Briefly, HCT116 and Mus81−/− HCT116 cells were pulsed with 100 μM of BrdU for 30 minutes, then harvested immediately (0h) or chased for 1h or 6h in pre-warmed fresh medium without BrdU. Cells were collected and the cell pellet was resuspended in cold PBS. An alkaline assay was performed according to the manufacture’s instruction (Trevigen, 4250-050-K). Following electrophoresis, the gels were neutralized by 0.4 M Tris.HCl and washed with PBS prior to immunostaining. The BrdU labeled nascent DNA were detected by mouse anti-BrdU antibody (Becton Dickinson, cat.347580, 1:10), then goat anti-mouse IgG conjugated with Alexa 488 (Invitrogen, A-21121, 1:100). The gels were then counterstained with DAPI. The samples were then dried according to the manufacture’s instruction (Trevigen, 4250-050-K). Images were taken by a BD pathway 855 microscope controlled by AttoVision (Becton Dickinson) with 10X or 20X objective lens. Images were analyzed by CometScore software (TriTeK).
8
Quantitative Analysis of RGC Morphology
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The analysis of RGC morphology was done by taking a mosaic of pictures at 10X (Pathway 855, Becton Dickinson). The number of RGCs, the number of neurites growing from each RGC (complexity), and the length of each neurite were measured and the cells were classified by length of the longest neurite in at least 3 wells/experimental condition in a total of three independent experiments.
For the retina and optic nerve, at least 3 sections were analysed from each of 3 rats for the analysis. To measure the in vitro expression of integrins, at least 3 coverslips per integrin and per substrate were analysed in at least three independent experiments. Analysis was performed using AttoVision (Beckton Dickinson)
For the retina and optic nerve, at least 3 sections were analysed from each of 3 rats for the analysis. To measure the in vitro expression of integrins, at least 3 coverslips per integrin and per substrate were analysed in at least three independent experiments. Analysis was performed using AttoVision (Beckton Dickinson)
9
BrdU Comet Assay for DNA Damage
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