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1

Infection and Quantification of Listeria in Bone Marrow-Derived Macrophages

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After 7–10 days of differentiation, BMDMs were washed twice and detached with ice-cold Versene buffer (0.8 mM EDTA, 1 mM glucose in PBS^−/−) for 20 min at 4°C. BMDMs were then seeded onto coverslips in 24-well tissue culture plates at 8.0 × 10⁵ cells/well to generate a monolayer. After 18 h, the monolayer was infected with wild type Lm at an MOI of 0.01 in RPMI-1640. At 30 min p.i., cells were washed three times with PBS and cultured in RPMI-1640 medium containing 10% FBS. At 60 min p.i., cells were washed three times with PBS and RPMI-1640 containing 10% FBS, 10% L929 conditioned medium, and 50 μg ml⁻¹ gentamycin was added to the cultures. At 18 h p.i., cells were fixed with 2.5% paraformaldehyde for 30 min at 37°C and prepared for fluorescence microscopy. The number of infected cells per foci-of-infection was quantified using epifluorescence microscopy. Images for analysis were taken with a Hamamatsu Orca R2 camera and Nikon Ti-E microscope with 10X objective and Volocity software (Improvision).

Czuczman M.A., Fattouh R., van Rijn J., Canadien V., Osborne S., Muise A.M., Kuchroo V.K., Higgins D.E, & Brumell J.H. (2014). Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread. Nature, 509(7499), 230-234.

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Infection and Quantification of Listeria in Bone Marrow-Derived Macrophages

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After 7–10 days of differentiation, BMDMs were washed twice and detached with ice-cold Versene buffer (0.8 mM EDTA, 1 mM glucose in PBS^−/−) for 20 min at 4°C. BMDMs were then seeded onto coverslips in 24-well tissue culture plates at 8.0 × 10⁵ cells/well to generate a monolayer. After 18 h, the monolayer was infected with wild type Lm at an MOI of 0.01 in RPMI-1640. At 30 min p.i., cells were washed three times with PBS and cultured in RPMI-1640 medium containing 10% FBS. At 60 min p.i., cells were washed three times with PBS and RPMI-1640 containing 10% FBS, 10% L929 conditioned medium, and 50 μg ml⁻¹ gentamycin was added to the cultures. At 18 h p.i., cells were fixed with 2.5% paraformaldehyde for 30 min at 37°C and prepared for fluorescence microscopy. The number of infected cells per foci-of-infection was quantified using epifluorescence microscopy. Images for analysis were taken with a Hamamatsu Orca R2 camera and Nikon Ti-E microscope with 10X objective and Volocity software (Improvision).

Czuczman M.A., Fattouh R., van Rijn J., Canadien V., Osborne S., Muise A.M., Kuchroo V.K., Higgins D.E, & Brumell J.H. (2014). Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread. Nature, 509(7499), 230-234.

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Quantifying Coumarin-6 NP Uptake Kinetics

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To quantitate uptake kinetics, coumarin-6 NPs were prepared for observation with confocal microscopy. T cells were seeded at 1 × 10³ cells per well and incubated with coumarin-6 NPs suspended in medium with 5 μM rhodamine at 37 °C with 5 % CO₂. At various time points (from 30 min to 6 h), wells were treated with Hoechst 33342 nucleic acid stain (Invitrogen) for 15 min. The medium was removed, and cells were washed three times with PBS and fixed with methanol for 25 min. Confocal fluorescence images were acquired with a Nikon Ti-E microscope equipped with an UltraVIEW VoX confocal attachment (Perkin Elmer).

Tang X., Liang Y., Liu X., Zhou S., Liu L., Zhang F., Xie C., Cai S., Wei J., Zhu Y, & Hou W. (2015). PLGA-PEG Nanoparticles Coated with Anti-CD45RO and Loaded with HDAC Plus Protease Inhibitors Activate Latent HIV and Inhibit Viral Spread. Nanoscale Research Letters, 10, 413.

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Live-cell Imaging Techniques Protocol

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Live-cell time lapse, live-cell imaging and fluorescence recovery after photobleaching (FRAP) experiments were performed with a Nikon Ti-E microscope equipped with an UltraVIEW VoX spinning disk confocal unit (PerkinElmer, UK), controlled by Volocity 6.3 software (PerkinElmer, UK), and equipped with a live-cell chamber (ACU control, Olympus) set at 37 °C with 5% CO₂ and 60% air humidity. Z-stacks were acquired with a ×60/1.49 NA CFI Apochromat TIRF oil immersion objective (voxel size, 0.12 × 0.12 × 0.3–1 µm; Nikon, Tokyo, Japan) or a 100x/1.49 NA CFI Apochromat TIRF oil immersion objective (voxel size, 0.071 × 0.071 × 0.5–1 µm; Nikon, Tokyo, Japan) and a cooled 14-bit CCD camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan, Cat.No.: C9100-50). Z-stack images were analyzed using Volocity 6.3 (PerkinElmer, UK) and Fiji. Mid Z-planes were assembled onto videos and annotated using Fiji^{98 (link)} (https://Fiji.nih.gov/ij/).

Arroyo M., Hastert F.D., Zhadan A., Schelter F., Zimbelmann S., Rausch C., Ludwig A.K., Carell T, & Cardoso M.C. (2022). Isoform-specific and ubiquitination dependent recruitment of Tet1 to replicating heterochromatin modulates methylcytosine oxidation. Nature Communications, 13, 5173.

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5

Immunofluorescence Imaging of β-Catenin and Lamp1

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The following antibodies were used for immunofluorescence (IF): anti-β-catenin (1:100; C2206; Sigma-Aldrich), anti-Lamp1 (1:100; 555798; BD Biosciences), Alexa Fluor 568 goat anti-mouse secondary (1:500; A-11031; Life Technologies), and Alexa Fluor 488 goat anti-rabbit secondary (1:500; A-11034; Life Technologies). IF was performed on cells cultured on glass-bottom dishes (P35G-1.5-20-C; MatTek), as described previously (Overholtzer et al., 2007 (link)). Briefly, cells were fixed in 1:1 methanol/acetone for 5 min at −20°C, followed by three 5-min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 hr, followed by incubation with primary antibodies at 4°C overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (1:1,000; D1306; Life Technologies). Confocal microscopy was performed with the Ultraview Vox spinning-disk confocal system (PerkinElmer) equipped with a Yokogawa CSU-X1 spinning-disk head and an electron-multiplying charge-coupled device camera (Hamamatsu C9100-13) coupled to a Nikon Ti-E microscope; image analysis was done using Volocity software (PerkinElmer).

Hamann J.C., Surcel A., Chen R., Teragawa C., Albeck J.G., Robinson D.N, & Overholtzer M. (2017). Entosis Is Induced by Glucose Starvation. Cell reports, 20(1), 201-210.

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Analyzing PML and Sp100A Colocalization

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Cells grown on glass coverslips were transfected and/or infected. Immunofluorescence experiments were performed using previously described protocols (78 (link)). Digital images were acquired using a Nikon TiE microscope equipped with the Perkin-Elmer UltraView Vox system. The colocalization of PML and Sp100A was analyzed by determining Pearson’s correlation coefficient. Average minimal distances between PML tracks and RCs were measured in two dimensions (2D) in 154 cells infected with HAdV wt and in 154 cells infected with the HAdV E2A SCM mutant. Pictures were analyzed with Volocity 6.2.1 software (Perkin-Elmer). Images were cropped using Adobe Photoshop CS6 and assembled with Adobe Illustrator CS6.

Stubbe M., Mai J., Paulus C., Stubbe H.C., Berscheminski J., Karimi M., Hofmann S., Weber E., Hadian K., Hay R., Groitl P., Nevels M., Dobner T, & Schreiner S. (2020). Viral DNA Binding Protein SUMOylation Promotes PML Nuclear Body Localization Next to Viral Replication Centers. mBio, 11(2), e00049-20.

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7

Immunofluorescence Imaging of β-Catenin and Lamp1

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The following antibodies were used for immunofluorescence (IF): anti-β-catenin (1:100; C2206; Sigma-Aldrich), anti-Lamp1 (1:100; 555798; BD Biosciences), Alexa Fluor 568 goat anti-mouse secondary (1:500; A-11031; Life Technologies), and Alexa Fluor 488 goat anti-rabbit secondary (1:500; A-11034; Life Technologies). IF was performed on cells cultured on glass-bottom dishes (P35G-1.5-20-C; MatTek), as described previously (Overholtzer et al., 2007 (link)). Briefly, cells were fixed in 1:1 methanol/acetone for 5 min at −20°C, followed by three 5-min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 hr, followed by incubation with primary antibodies at 4°C overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (1:1,000; D1306; Life Technologies). Confocal microscopy was performed with the Ultraview Vox spinning-disk confocal system (PerkinElmer) equipped with a Yokogawa CSU-X1 spinning-disk head and an electron-multiplying charge-coupled device camera (Hamamatsu C9100-13) coupled to a Nikon Ti-E microscope; image analysis was done using Volocity software (PerkinElmer).

Hamann J.C., Surcel A., Chen R., Teragawa C., Albeck J.G., Robinson D.N, & Overholtzer M. (2017). Entosis Is Induced by Glucose Starvation. Cell reports, 20(1), 201-210.

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Ti e microscope

Lab products found in correlation

7 protocols using ti e microscope

Infection and Quantification of Listeria in Bone Marrow-Derived Macrophages

Infection and Quantification of Listeria in Bone Marrow-Derived Macrophages

Quantifying Coumarin-6 NP Uptake Kinetics

Live-cell Imaging Techniques Protocol

Immunofluorescence Imaging of β-Catenin and Lamp1

Analyzing PML and Sp100A Colocalization

Immunofluorescence Imaging of β-Catenin and Lamp1

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