Cell culture reagents were obtained from Hyclone (GE, Boston, MA, USA) unless otherwise noted. MDA-MB-231 breast tumor cells were obtained from the American Type Culture Collection and maintained in Dulbecco′s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum. For imaging assays, cells were plated in 12-well plates (Corning, Corning, NY, USA) and allowed to grow to ~50% confluency before being treated with solutions of SPcs in media (1% dimethylsulfoxide). Images were collected with a VWR Inverted Fluorescence Microscope, Moticam 5.0 (Motic, Richmond, BC, Canada), and TRITC filter set (λex = 540 nm ± 12 nm, λem = 605 nm ± 27 nm).
Inverted fluorescence microscope
Manufactured by Motic
Sourced in China
The Inverted Fluorescence Microscope is a laboratory instrument designed to examine fluorescent samples. It features an optical system that illuminates the specimen from below, allowing for the observation of cells or tissues cultured on slides or in petri dishes. The microscope utilizes specialized filters and light sources to excite fluorescent molecules within the sample, enabling the visualization of specific cellular structures or biological processes.
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Lab products found in correlation
Tunel apoptosis assay kit, by Beyotime (2 mentions)
Antifade mounting medium, by Beyotime (2 mentions)
Paraformaldehyde (pfa), by Beyotime (2 mentions)
Cell culture reagents, by GE Healthcare (1 mentions)
Moticam 5, by Motic (1 mentions)
12 well plate, by Corning (1 mentions)
Mda mb 231 breast tumor cells, by American Type Culture Collection (1 mentions)
Dapi solution, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Sangon (1 mentions)
Triton x 100, by Sangon (1 mentions)
Tunel kit, by Beyotime (1 mentions)
Tunel apoptosis kit, by Beyotime (1 mentions)
Triton x 100, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Sangon (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Cell counting kit 8 (cck8), by Sangon (1 mentions)
Cell cycle analysis kit, by Beyotime (1 mentions)
5 protocols using inverted fluorescence microscope
1
Fluorescence Imaging of Breast Cancer Cells
2
Apoptosis Evaluation via TUNEL Assay
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At 48 h after transfection, the TUNEL Apoptosis Assay Kit (Beyotime, China) was applied to assess cell apoptosis. In brief, first the cells were fixed at room temperature with 10% neutral formalin (Sangon, China) for 30 minutes (min). Secondly, the cells were incubated with TUNEL working solution in the dark at 37°C for 1 h. Thirdly, the images were captured using an inverted fluorescence microscope (Motic, China).
3
Cell Proliferation and Apoptosis Assay
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To evaluate cell proliferation, 10 µl Cell Counting Kit-8 reagent (Sangon Biotech Co., Ltd.) was added to the 3×103 cells at 0, 24, 48 and 72 h post-transfection. The cells were then cultured at 37°C for 2 h, and the optical density value at 450 nm was measured. Cell apoptosis was evaluated using a TUNEL kit (Beyotime Institute of Biotechnology) at 48 h post-transfection. The 6×104 cells were cultured with TUNEL working solution at 37°C in the dark for 1 h after being fixed in 4% paraformaldehyde (Sangon Biotech Co., Ltd.) at room temperature for 0.5 h and permeabilized with 0.3% Triton X-100 (Sangon Biotech Co., Ltd.) for 5 min at room temperature. The cells were stained with DAPI solution (Beyotime Institute of Biotechnology) for 5 min at 37°C and antifade Mounting Medium (Beyotime Institute of Biotechnology) before being sealed. The images of 5 fields of view were captured by an inverted fluorescence microscope (Motic Incorporation, Ltd.).
4
TUNEL Apoptosis Assay in HemECs
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A TUNEL apoptosis kit (Beyotime Institute of Biotechnology) was used for detecting the apoptosis of HemECs following transfection. Briefly, transfected HemECs were plated into 24-well plates (1x104 cells/well) and cultured for 48 h. The HemECs were then fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology) for 30 min at 37˚C and incubated with 0.1% Triton X-100 (Beyotime Institute of Biotechnology) for 5 min at 4˚C. Subsequently, 50 µl TUNEL solution was added and the cells were incubated for 1 h at 37˚C, followed by DAPI (Sangon Biotech Co., Ltd.) staining for 15 min at room temperature. After adding Antifade Mounting Medium (Beyotime Institute of Biotechnology), the cells were sealed and observed under an inverted fluorescence microscope (Motic China Group Co., Ltd.) with 3 random fields being chosen.
5
Comprehensive Cell Characterization Assay
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The cell viability was detected by cell counting kit−8 (Sangon, China). About 10 μl CCK−8 reagent mixing with 100 μl RPMI 1640 medium was added and incubated with cells for 2 h at 37°C. Then a microplate reader (BioTek, USA) was adopted to read optical density value. The cell apoptosis determination was conducted by TUNEL Apoptosis Assay Kit (Beyotime, China). In brief, the cells were fixed with 4% paraformaldehyde (Beyotime, China) for 30 min at room temperature. Then the cells were incubated with working solution at 37°C for 1 h. An inverted fluorescence microscope (Motic, China) was applied to capture images. Cell Cycle Analysis Kit (Beyotime, China) was applied to complete cell cycle analysis and the kit’s protocol was strictly followed. A flow cytometer (BD, USA) was used to determine the cells, and data were analyzed by Flowjo 7.6 (BD, USA).
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