Dmi 3000 fluorescent microscope

Manufactured by Leica

The Leica DMI 3000 is a fluorescent microscope designed for high-quality imaging and analysis. It features a LED illumination system, motorized components, and advanced optics for capturing detailed fluorescent images. The microscope is suitable for a variety of applications that require fluorescent observation and documentation.

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Lab products found in correlation

A1rmp confocal microscope, by Nikon (2 mentions) Click it plus edu alexa fluor 555 imaging kit, by Thermo Fisher Scientific (1 mentions) Cfi apo lwd objective, by Nikon (1 mentions) Mouse on mouse m o m basic kit, by Vector Laboratories (1 mentions) Antigen unmasking solution, by Vector Laboratories (1 mentions) Synergy ht plate, by Agilent Technologies (1 mentions)

5 protocols using dmi 3000 fluorescent microscope

Assessing Cell Proliferation Dynamics

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Three thousand cells per well were seeded as in the three-dimensional cell culture protocol above. On Day 8, 24 hours after a proteasome inhibitor treatment, cells were rinsed once with PBS, and replaced with fresh cell culture media (10% FBS). The media were refreshed on Day 9 and then every three days. On Day 16, cells were counterstained with Hoechst 33342 and fixed with PFA or stayed in fresh cell culture media. Images were taken using a Leica DMI 3000 fluorescent microscope with a 10X objective. For each condition, images were taken and quantified from four biological replicates. The total number of nuclei of each spheroid was counted. On Day 16 (end of the experiments), spheroids with a greater number of nuclei than that of the average plus standard deviation in the cytostatic state (Day 7) were scored as regrowth. Statistical significance was determined by two-tailed Student’s t-tests.

Kim L.M., Kim P.Y., Gebreyohannes Y.K, & Leung C.T. (2022). Sustained oncogenic signaling in the cytostatic state enables targeting of non-proliferating persistent cancer cells. Cancer research, 82(17), 3045-3057.

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Click-iT EdU Alexa Fluor 555 Imaging for Cell Cycle Analysis

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Click-iT^® Plus EdU Alexa Fluor^® 555 Imaging Kit (Invitrogen) was used. Six days after vehicle (water or DMSO) or a cytostatic drug (palbociclib or lapatinib) treatment, EdU reagent was added together with vehicle or the cytostatic drug for 24 hours. Cells were then fixed with 4% PFA and processed following the 3D immunofluorescence staining protocol. After TritonX-100, glycine and IF buffer washes, Click-iT^® reaction was performed according to vendor protocol. Samples were counterstained with DAPI, mounted with Prolong Diamond Antifade reagent, and analyzed using Leica DMI 3000 fluorescent microscope with 20X objective. Images were acquired using a Nikon A1RMP confocal microscope with a 20X Objective. Statistical significance was determined by two-tailed Student’s t-tests.

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TUNEL Assay for Apoptosis Detection

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Click-iT^® Plus TUNEL Alexa^® 594 In Situ Apoptosis Detection Kit (Invitrogen) was used. Twenty-four hours after bortezomib treatment, cells were rinsed once with PBS, fixed with 4% PFA, and processed following the 3D immunofluorescence staining protocol. After TritonX-100, glycine and IF buffer washes, cells were washed twice in deionized water and incubated with the TdT Reaction buffer for 10 minutes at room temperature. Samples were then incubated with the TdT Reaction mixture overnight in a humidified chamber at room temperature. Cells were washed in IF buffer three times for 20 minutes each, once with PBS and processed with the Click-iT Plus TUNEL reaction cocktails for 30 minutes at room temperature in the dark. Samples were counterstained with DAPI and mounted with Prolong Diamond Antifade reagent. Samples were analyzed using a Leica DMI 3000 fluorescent microscope with a 20X objective. Images were acquired using a Nikon A1RMP confocal microscope with a 25X CFI Apo LWD Objective. Statistical significance was determined by two-way ANOVA.

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Quantifying Tumor Proliferation and Apoptosis

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Tissue sections (7 μm thick) were de-paraffinized and processed for antigen retrieval by heating in Antigen Unmasking Solution (Vector Laboratories) for 30 minutes. Tissue sections were stained using Mouse on Mouse (M.O.M.) Basic Kit (Vector Laboratories) according to the vendor protocol. Primary antibodies against Ki67 and cleaved caspase-3 were used. Alexa Fluor^® Plus 488 and DyLight 549 Streptavidin secondary antibodies were used. Slides were counterstained with DAPI and mounted with Prolong Diamond Antifade reagent. Images were taken using Leica DMI 3000 fluorescent microscope with a 20X objective. Four to six different tumors for each condition were analyzed using CellProfiler 3.1.8 (RRID:SCR_007358). Statistical significance was determined by two-tailed Student’s t-tests.

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Cell Viability Assay for Encapsulated Cells

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For the cell compatibility tests, 293T cells were used. When grown to 80% confluency, cells were trypsinized and seeded in 48-well plates at a density of 5 × 10 3 cells/well. The cells were cultured overnight, washed twice with warm PBS and treated overnight with 1 mL of the incubation medium withdrawn on selected time points. Upon removal of the incubation medium, cells were washed twice with warm PBS and 500 μL of MTT reagent (2.5 mg/mL) was added to sample wells. After 2 hours, the solution was aspirated, and 1 mL of DMSO was added to dissolve the formazan crystals. Absorbance was read at 570 nm using a BioTek Synergy HT plate reader.
To visualize the viability of encapsulated cells, cell-laden hydrogels were stained with Live/Dead cell double-staining kit which renders viable cells green (calcein AM) and dead cells red (ethidium). Dyes were prepared according to the manufacturer's protocol and added to sample wells for 20 minutes prior to washing and observation with a Leica DMI3000 fluorescent microscope.

Peng S., Lai Z.T., Hong D.W., Chu I.M, & Lai P.L. (2017). Controlled release of strontium through neutralization reaction within a methoxy(polyethylene glycol)-polyester hydrogel. Journal of applied biomaterials & functional materials, 15(2).

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