Fig 1: Tumor sphere formation of 5-FU-resistant HCT116RF10 and parental HCT116 cells. (a) Tumor sphere formation was analyzed using a Leica DMi1 microscope with 50× magnification. Scale bar = 500 µm. HCT116RF10 and parental HCT116 cells were treated with or without 3 × 10-5 M 5-FU for 3 or 14 days. Control, no 5-FU, solvent (DMSO) alone. To assess the ability of HCT116RF10 and parental HCT116 cells to form tumor spheres, the cells were treated with solvent alone (b) or 3 × 10-5 M 5-FU (c) for 14 days. Tumor sphere size was calculated as described in the Materials and Methods. White circle, HCT116 cells; black circle, HCT116RF10 cells. (d) Drug sensitivity of 5-FU in HCT116 and HCT116RF10 tumor spheres. Tumor sphere formation by HCT116RF10 and parental HCT116 cells after a 14-day treatment with 5-FU at the indicated concentrations. Results are the averages for groups of three tumor spheres each with error bars showing SE. White circle, HCT116 cells; black circle, HCT116RF10 cells.
Fig 2: a Microphotographs of hPDLSCs cultured in the presence of experimental conditions for 10 and 28 days. Images were obtained using an inverted microscope (Leica DMi1, Wetzlar, Germany). Scale bar: 20 µm. b MTT assay was performed on hPDLSCs in presence of no-OM, and exposed to ELF-EMF or sham system, for 6, 24 and 48 h. c Western blot analysis of Ki-67 expression. Data are expressed as mean ± SD of two experiments performed in triplicate. ***p < 0.001
Fig 3: Morphological observation of bone marrow-derived dendritic cells (BMDCs) during the differentiation procedures. The pictures were captured by inverted microscope equipped with a digital imaging system (Leica DMi1; Leica Microsystems, Germany). (A) BMDCs observed at day 2 of culture. (B) Morphology of the non-stimulated BMDCs (arrows) at day 7 of culture. (C) Morphology of DCs (arrows) pulsed with lipopolysaccharide at day 9 of culture. (D) Morphology of DCs (arrows) stimulated with Salmonella Typhi ghost at day 9 of culture. Scale bars = 50 µm (A–D).
Fig 4: GSK3ß is necessary for the necrotic response elicited by DMNQ.a Cytofluorimetric analysis measuring cell death percentages (PI positivity) in the different U87MG clones treated with the indicated concentrations of DMNQ for 24 h. Data are presented as mean ± SD. n = 3. b Cytofluorimetric analysis measuring cell death percentages (PI positivity). U87MG cells were transfected with siRNAs against GSK3ß or control. After 48 h they were treated with 30 µM of DMNQ for further 24 h. Data are presented as mean ± SD. n = 3. c Immunoblot analysis of GSK3ß levels in U87MG cells transfected with siRNAs against GSK3ß or control. Actin was used as loading control. d Immunoblot analysis of GSK3ß levels in U87MG GSK3ß+/+ and GSK3ß-/- cells retrovirally infected with the GSK3ß-GFP fusions WT or its catalytically inactive mutant K85A (KM). Actin was used as loading control. e Cytofluorimetric analysis measuring cell death percentages (PI positivity) in the indicated U87MG cell lines treated with 30 µM of DMNQ for 24 h. Data are presented as mean ± SD. n = 3. f Immunoblot analysis of Caspase-3, Caspase-2, and HDAC4 caspase-dependent processing in the indicated U87MG cell lines treated with 30 µM of DMNQ. Incubation with the combination TRAIL (2.5 ng/ml) and bortezomib (0.1 µM) for 20 h was used to trigger apoptosis. Actin was used as loading control. g Cytofluorimetric analysis measuring cell death percentages (PI positivity) in the indicated U87MG cell lines treated or not with the combination TRAIL (2.5 ng/ml) and bortezomib (0.1 µM) for 24 h. Data are presented as mean ± SD. n = 3. h Microscopic images of the indicated U87MG cell lines treated for 24 h with DMNQ (30 µM). Arrows point to membrane blistering and arrowheads to examples of necrotic cells. Phase contrast images were obtained with Leica DMi1 microscope with a 10x objective. i qRT-PCR analysis of GSK3ß mRNA levels in the silenced IMR90-E1A//BCL2/C9DN cells. Data are from three experiments; +SD. j Cytofluorimetric analysis measuring cell death percentages (PI positivity). IMR90-E1A/BCL2/C9DN cells were transfected with siRNAs against GSK3ß or control and after 48 h treated with 100 µM of DMNQ for further 24 h. Data are presented as mean ± SD. n = 3.
Fig 5: Cell migration - scratch assay: collection of microscope (Leica DMi1; inverted phase contrast) images (4X) concerning day 0 and day 5 after treatment with and without (Control) complex 2 R .
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