M. tuberculosis cultures were grown to exponential phase (OD600 −0.5) and then treated with 10× MIC of the indicated antibiotic. Aliquots were taken after 48 hours of treatment, washed once in PBS + 0.05% Tween 80 (PBST) and fixed in 4% paraformaldehyde for 2 hours at room temperature. The cells were washed once, resuspended in PBST and stained with FM4–64FX (Thermo Fisher Scientific) at a final concentration of 1 μg ml−1 for 30 minutes in the dark at room temperature. Once stained, 1 μl of cells was spotted onto a 1.5% low-melting agarose pad and observed with a Nikon Ti2-E inverted fluorescence microscope using a ×100 oil immersion objective lens. Images were acquired by NIS-Elements at a resolution of 2,048 × 2,048 and processed with Fiji software39 (link). Segmentation and calculation of cellular length from these images was done using the plug-in MicrobeJ40 (link).
Ti2 e inverted fluorescence microscope
Manufactured by Nikon
The Ti2-E inverted fluorescence microscope is a high-performance laboratory equipment designed for advanced imaging applications. It features a robust and versatile optical system that enables the observation and capture of fluorescence signals from samples. The microscope's core function is to provide users with the ability to visualize and analyze fluorescently labeled specimens with precision and clarity.
Automatically generated - may contain errors
Lab products found in correlation
Fm4 64fx, by Thermo Fisher Scientific (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Elements high content imaging software, by Nikon (1 mentions)
μ slide 8 well plate, by Ibidi (1 mentions)
C fll lfov, by Nikon (1 mentions)
Ds qi2 cmos camera, by Nikon (1 mentions)
5 protocols using ti2 e inverted fluorescence microscope
1
Microscopic analysis of M. tuberculosis
2
Passaging and Imaging hMSCs in PEG-MMP Scaffolds
3
Quantifying Polyomavirus Infection via Immunofluorescence
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To score infections at indicated time points, complete media was aspirated, and cells were fixed in ice-cold methanol for at least 20 min at −20°C. Cells were rehydrated with 1 × PBS, further permeabilized with 0.1% Triton-X for 10 min at room temperature, and blocked with 5% goat serum (ThermoFisher) and 0.1% Triton-X (USB) in 1 × PBS for 1 h at room temperature. Cells were incubated with the VP1-specific primary antibody PAb597 in 1 × PBS (diluted 1:50) overnight at 4°C, washed extensively with 1 × PBS, and then incubated with goat anti-mouse Alexa Fluor 488 conjugated antibody (ThermoFisher) in 1 × PBS (diluted 1:1,000) for 1 h at room temperature in the dark. Finally, cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) in 1 × PBS (diluted 1:1000) for 5 min at room temperature, and washed again in 1 × PBS prior to imaging. The 20x objective of a Ti2-E inverted fluorescence microscope (Nikon) was used to analyze cells for nuclear VP1 staining and calculate total cell number. Cell count analysis was performed using Elements High Content imaging software (Nikon). PAb597 was generated against SV40 VP1 but also cross-reacts with BKPyV VP1 and JCPyV VP1, which enables the efficient scoring of human and simian polyomavirus infection with 1 primary antibody. The PAb957 hybridoma was a generous gift from Ed Harlow.
4
Multicolor Virus Imaging in PK15 Cells
5
High-content Fluorescence Microscopy Imaging
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Microscopy imaging was performed using a Nikon Ti-2E inverted fluorescence microscope. Images were acquired using a Nikon CFI Plain Fluor 10× objective and a 14-bit Nikon DS-Qi2 CMOS camera. Images were captured using four channels: brightfield with phase contrast, DAPI (Nikon C-FLL LFOV, 392/23 nm excitation, 447/60 nm emission and 409 nm dichroic mirror), mCherry (Nikon C-FLL LFOV, 562/40 nm excitation, 641/75 nm emission and 593 dichroic mirror) and EGFP (Nikon C-FLL LFOV, 466/40 nm excitation, 525/50 nm emission and 495 nm dichroic mirror). Illumination for brightfield imaging was performed using the built in Ti-2E LED. Epifluorescence excitation was performed using a 130 W mercury lamp (Nikon C-HGFI). Gain, exposure and vertical offset were automatically determined using built-in NIS functions to avoid user biasing. Cells were imaged in Greiner Sensoplate 96-well glass bottom multiwell plates (M4187-16EA, Sigma-Aldrich). The concentration of cells in each well were diluted down to ~1000 cells to ensure adequate spacing between adjacent cells. An automated procedure was run on NIS using the Jobs function to take 16 images, on each of the 4 channels, inside of each well. The images were exported from NIS to standard TIFF format.
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