PBMCs from LTBI individuals were infected with M.tb H37Rv (MOI 1:1) on glass coverslips in 24-well tissue culture plates as previously described [27 (link)]. Robust, granuloma-like structures were visually confirmed at 7 days post-infection and fixed with 4% paraformaldehyde. The structures were washed and incubated overnight at 4°C with a 30 nM solution of cFLFLFK-PEG12-Cy3 diluted in HBSS+. The next day, granulomas were washed three times with PBS, blocked with 10% goat serum and 5% bovine serum albumin in PBS for 1 h, and incubated with a mouse anti-human CD68 antibody (DAKO) for 1 h. Goat anti-mouse AF647 (Invitrogen Life Technologies, Carlsbad, CA) secondary antibody was applied for 1 h after additional washes. Cell nuclei were stained with 400 ng/ml DAPI DNA stain (Molecular Probes, Carlsbad, CA). Finally, the coverslips were washed three times with distilled water and mounted on glass slides using ProLong Gold Antifade (Invitrogen Life Technologies). All incubations were performed at room temperature. Images of peptide binding and CD68 expression on granulomas were captured on an FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan) using a 60× oil DIC objective.
Dapi dna stain
Manufactured by Thermo Fisher Scientific
DAPI is a fluorescent DNA stain commonly used in microscopy and flow cytometry. It binds to the minor groove of DNA, allowing for the visualization and quantification of DNA content within cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade, by Thermo Fisher Scientific (2 mentions)
Mouse anti human cd68 antibody, by Agilent Technologies (1 mentions)
Goat anti mouse af647, by Thermo Fisher Scientific (1 mentions)
Fv3000 confocal laser scanning microscope, by Olympus (1 mentions)
Tma dph, by Thermo Fisher Scientific (1 mentions)
Fm4 64, by Thermo Fisher Scientific (1 mentions)
Nile red dye, by Merck Group (1 mentions)
Fluoview 10i, by Olympus (1 mentions)
Ti e microscope, by Nikon (1 mentions)
Fm4 64 membrane stain, by Thermo Fisher Scientific (1 mentions)
4 protocols using dapi dna stain
1
Visualizing Granuloma Formation and Peptide Binding
2
Microscopic Imaging of Fluorescently Labeled Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Culture samples (1 ml) were collected and pelleted by centrifugation at room temperature at 6,010 x g in a tabletop microfuge. The supernatants were removed using aspiration and resuspended in ~10 μl PBS containing the indicated dyes at the following final concentrations: 2.0 μg/ml DAPI DNA stain (Molecular Probes); 0.02 mM TMA-DPH (Life Technologies) or 3.0 μg/ml FM4-64 (Life Technologies). After resuspension, samples were mounted on glass slides with polylysine-treated coverslips. Exposure times were generally 1 sec. Images were collected with a Nikon Ti-E microscope equipped with a CFI Plan Apo lambda DM 100X objective, and Prior Scientific Lumen 200 Illumination system, C-FL UV-2E/C DAPI, and C-FL Texas Red HC HISN Zero Shift filter cubes, and a CoolSNAP HQ2 monochrome camera. All obtained images were captured with NIS Elements Advanced Research (version 4.10), and processed with ImageJ64 [39 ].
3
Confocal Imaging of Granuloma-like Structures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Granuloma-like structures were formed on glass coverslips in 24-well tissue culture plates. Cells were stained with Nile red dye (Sigma-Aldrich, St. Louis, Mo), incubated in the dark for 15 min, and washed three times with distilled water. Counterstaining was performed with 0.1% potassium permanganate, and coverslips were washed three times with distilled water. Finally, 0.1 µg/ml of DAPI DNA stain (Molecular Probes, Carlsbad, CA)–PBS was added for 5 min. The coverslips were then washed three times with distilled water and mounted on glass slides using ProLong Gold Antifade (Invitrogen, Carlsbad, CA). Each sample was assessed by confocal microscopy (Olympus Fluoview 10i). At least 15 separate high-power fields per sample were evaluated, and at least 3 replicates were analyzed.
4
Fluorescence Microscopy of Bacterial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All samples were grown in CH media overnight at room temperature to mid-exponential, back-diluted to OD 600 5 0.008 in 25 ml CH, and grown at 378C in a shaking waterbath set at 280 rpm for 1.5 h. When indicated, 1 mM IPTG was added, and cells were grown for an additional 1.5 h. All cells were in mid-exponential growth when images were captured. To capture images, 1 ml of cells were pelleted at 6,010 3 g for 1 min in a tabletop microfuge at room temperature. The supernatant was removed by aspiration and the pellet resuspended in 10 ul PBS containing FM4-64 membrane stain (3 mg/ml) (Life Technologies) and DAPI DNA stain (2 mg/ml) (Molecular Probes). Cells were mounted on glass slides with polylysine treated coverslips immediately before imaging. Fluorescence microscopy was performed with a Nikon Ti-E microscope equipped with a CFI
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!