Deltavision ultra, by Cytiva - Product details

1

Immunofluorescence Assay for Cilia Analysis

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For immunofluorescence (IF) assays, cells were counted and seeded at 5×10⁴ cells on sterile glass coverslips. For assessing cilia, MEFs were serum-starved for 48 hours prior to analysis. Where indicated, drugs were added 24 hours prior to fixation. Cells were fixed in 4% PFA (Sigma Aldrich, St Louis, USA) or ice-cold Methanol (100%) for the centrosomal protein in PBS for 20 minutes at RT and permeabilized in 0.1% TritonX-100 (Sigma Aldrich) for 10 minutes or 90 seconds (cilia experiments) and blocked in 3% or 1% (cilia experiments) bovine serum albumin (BSA; Sigma Aldrich) in PBS for 1 hour in a humidified chamber at RT. Coverslips were washed and incubated with Alexa-fluor-conjugated secondary antibodies (Sigma Aldrich) diluted in 3 or 1% BSA (1:1000) for 30 minutes at 37°C in a humidified chamber in the dark. Coverslips were mounted using Prolong gold anti-fade mounting medium (Life Technologies). Imaging was performed on a DeltaVision personal DV deconvolution microscope and DeltaVision Ultra (super-resolution) (Applied Precision, GE Healthcare, Issaquah, WA) and analyzed using the GE DeltaVision software package. Automated counting was performed using script modules of Fiji-ImageJ software (Java3D, Minnesota, USA).

Rashidieh B., Shohayeb B., Bain A.L., Fortuna P.R., Sinha D., Burgess A., Mills R., Adams R.C., Lopez J.A., Blumbergs P., Finnie J., Kalimutho M., Piper M., Hudson J.E., Ng D.C, & Khanna K.K. (2021). Cep55 regulation of PI3K/Akt signaling is required for neocortical development and ciliogenesis. PLoS Genetics, 17(10), e1009334.

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2

Quantifying 53BP1 Foci in HOG Cells

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HOG-EV or HOG-R132H cells were plated at a density of 3 × 10⁴ cells per well onto 18 mm glass coverslips in 12-well plates. The following day, cells were treated with either DMSO or 10 nM BAY 2402234. Cells were fixed at the indicated time points with 4% formaldehyde, permeabilized with 0.3% Triton X-100 in PBS, incubated with Triton Block (0.2 M glycine, 2.5% FBS, 0.1% Triton X-100 in PBS), and immunostained with an anti-53BP1 antibody (1:1,000, Novus Biologicals, NB100–304) diluted in Triton Block. Cells were washed, incubated with a donkey anti-rabbit Alexa Fluor 488 secondary antibody (Invitrogen, A21206), and mounted. Images were acquired using a DeltaVision Ultra (Cytiva) microscope equipped with a 60× objective with 9× 0.5 μm z sections. Images were deconvolved and maximum intensity projections were generated. 53BP1 foci were quantified in individual cells using ImageJ with a macros plugin (n = 325–505 cells per condition per time point).

Shi D.D., Savani M.R., Levitt M.M., Wang A.C., Endress J.E., Bird C.E., Buehler J., Stopka S.A., Regan M.S., Lin Y.F., Puliyappadamba V.T., Gao W., Khanal J., Evans L., Lee J.H., Guo L., Xiao Y., Xu M., Huang B., Jennings R.B., Bonal D.M., Martin-Sandoval M.S., Dang T., Gattie L.C., Cameron A.B., Lee S., Asara J.M., Kornblum H.I., Mak T.W., Looper R.E., Nguyen Q.D., Signoretti S., Gradl S., Sutter A., Jeffers M., Janzer A., Lehrman M.A., Zacharias L.G., Mathews T.P., Losman J.A., Richardson T.E., Cahill D.P., DeBerardinis R.J., Ligon K.L., Xu L., Ly P., Agar N.Y., Abdullah K.G., Harris I.S., Kaelin WG J.r, & McBrayer S.K. (2022). De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma. Cancer cell, 40(9), 939-956.e16.

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3

Quantifying 53BP1 Foci in HOG Cells

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HOG-EV or HOG-R132H cells were plated at a density of 3 × 10⁴ cells per well onto 18 mm glass coverslips in 12-well plates. The following day, cells were treated with either DMSO or 10 nM BAY 2402234. Cells were fixed at the indicated time points with 4% formaldehyde, permeabilized with 0.3% Triton X-100 in PBS, incubated with Triton Block (0.2 M glycine, 2.5% FBS, 0.1% Triton X-100 in PBS), and immunostained with an anti-53BP1 antibody (1:1,000, Novus Biologicals, NB100–304) diluted in Triton Block. Cells were washed, incubated with a donkey anti-rabbit Alexa Fluor 488 secondary antibody (Invitrogen, A21206), and mounted. Images were acquired using a DeltaVision Ultra (Cytiva) microscope equipped with a 60× objective with 9× 0.5 μm z sections. Images were deconvolved and maximum intensity projections were generated. 53BP1 foci were quantified in individual cells using ImageJ with a macros plugin (n = 325–505 cells per condition per time point).

Shi D.D., Savani M.R., Levitt M.M., Wang A.C., Endress J.E., Bird C.E., Buehler J., Stopka S.A., Regan M.S., Lin Y.F., Puliyappadamba V.T., Gao W., Khanal J., Evans L., Lee J.H., Guo L., Xiao Y., Xu M., Huang B., Jennings R.B., Bonal D.M., Martin-Sandoval M.S., Dang T., Gattie L.C., Cameron A.B., Lee S., Asara J.M., Kornblum H.I., Mak T.W., Looper R.E., Nguyen Q.D., Signoretti S., Gradl S., Sutter A., Jeffers M., Janzer A., Lehrman M.A., Zacharias L.G., Mathews T.P., Losman J.A., Richardson T.E., Cahill D.P., DeBerardinis R.J., Ligon K.L., Xu L., Ly P., Agar N.Y., Abdullah K.G., Harris I.S., Kaelin WG J.r, & McBrayer S.K. (2022). De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma. Cancer cell, 40(9), 939-956.e16.

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4

Visualizing Aurora Kinase Inhibition

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Cells on glass bottom plates were incubated in 0.1 μg/mL hoescht for >30 minutes. For Aurora kinase inhibition experiments, cells on glass bottom plates were treated with 10 μM ZM447439 at 37°C for 2.5 hours. In the last 30 minutes before completing ZM447439 treatment, 0.1 μg/mL hoescht was added to the media. Images were taken on the Deltavision Ultra (Cytiva) system using a 60x/1.42NA objective. 8 μm images were taken with z-sections of 0.2 μm. All images presented were deconvolved and max projected. Images presented within the same panel were taken on the same day, camera settings, and scaled equivalently. Images with morphological markers such as DNA or tubulin were not all scaled equivalently.

Ly J., Blengini C.S., Cady S.L., Schindler K, & Cheeseman I.M. (2024). A conserved germline-specific Dsn1 alternative splice isoform supports oocyte and embryo development. bioRxiv.

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5

Hoescht Staining and Imaging GFP

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Cells on glass bottom plates were incubated in 0.1 μg/mL hoescht for >30 minutes. The GFP transgene was induced by addition of 1 μg/mL dox for 24 hours prior to imaging. Images were taken on the Deltavision Ultra (Cytiva) system using a 60x/1.42NA objective. 8 μm images were taken with z-sections of 0.2 μm. All images presented were deconvolved and max projected.

Ly J., Xiang K., Su K.C., Sissoko G.B., Bartel D.P, & Cheeseman I.M. (2024). Nuclear release of eIF1 globally increases stringency of start-codon selection to preserve mitotic arrest physiology. bioRxiv.

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6

High-Resolution Imaging of Interphase and Metaphase Cells

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Immunofluorescence images were captured on a DeltaVision Ultra (Cytiva) microscope system equipped with a 4.2 Mpx sCMOS detector. Interphase nuclei and micronuclei images were acquired with a ×100 objective (UPlanSApo, 1.4 NA) and 1 × 0.2 μm z-section. Quantitative fluorescence image analyses were performed using Fiji (v.2.1.0/1.53c). IF–FISH images were acquired with a ×60 objective (PlanApo N 1.42 oil) and 15 × 0.2 μm z-sections. Deconvolved maximum intensity projections were generated using softWoRx (v.7.2.1, Cytiva).
Metaphase FISH images were acquired on the Metafer Scanning and Imaging Platform microscope (Metafer 4, v.3.13.6, MetaSystems). The slides were first scanned for metaphases using M-search with a ×10 objective (ZEISS Plan-Apochromat 10x/0.45), and metaphases were automatically imaged using Auto-cap with a ×63 objective (ZEISS Plan-Apochromat 63x/1.40 oil). Images were analysed using the Isis Fluorescence Imaging Platform (MetaSystems) and Fiji (v.2.1.0/1.53c).

Lin Y.F., Hu Q., Mazzagatti A., Valle-Inclán J.E., Maurais E.G., Dahiya R., Guyer A., Sanders J.T., Engel J.L., Nguyen G., Bronder D., Bakhoum S.F., Cortés-Ciriano I, & Ly P. (2023). Mitotic clustering of pulverized chromosomes from micronuclei. Nature, 618(7967), 1041-1048.

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7

Chromosome Inheritance Tracking in Mitosis

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DLD-1 cells were seeded in four-well chamber slides and treated with or without DOX/IAA for 48 h. Cells were then arrested in G2 with 10 µM CDK1 inhibitor RO-3306 (Millipore-Sigma) for 16 h, washed with PBS three times and released into mitosis in fresh medium. After 90 min, cells were fixed with 4% formaldehyde followed by IF–FISH, as described above, and hybridized to X- and Y-chromosome paint probes (MetaSystems). For analysis of chromosome inheritance between daughter cells, pairs of newly formed daughter cells were imaged on the DeltaVision Ultra (Cytiva) microscope system. Images were split into separate channels for quantification using the ImageJ plugin Segmentation (Robust Automatic Threshold Selection) to create a mask for the FISH signals. Particles of the mask were analysed to generate a list of regions of interest for intensity measurements. FISH signal intensities were then measured in each pair of daughter cells for both the X and Y chromosomes. The distribution of FISH signal was calculated by the ratio of the daughter cell with the lower signal compared to the daughter cell with the higher signal.

Lin Y.F., Hu Q., Mazzagatti A., Valle-Inclán J.E., Maurais E.G., Dahiya R., Guyer A., Sanders J.T., Engel J.L., Nguyen G., Bronder D., Bakhoum S.F., Cortés-Ciriano I, & Ly P. (2023). Mitotic clustering of pulverized chromosomes from micronuclei. Nature, 618(7967), 1041-1048.

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Deltavision ultra

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7 protocols using deltavision ultra

Immunofluorescence Assay for Cilia Analysis

Quantifying 53BP1 Foci in HOG Cells

Quantifying 53BP1 Foci in HOG Cells

Visualizing Aurora Kinase Inhibition

Hoescht Staining and Imaging GFP

High-Resolution Imaging of Interphase and Metaphase Cells

Chromosome Inheritance Tracking in Mitosis

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