PHCs were seeded at 20,000 cells/cm2 on 6-well Matrigel-coated plates with HGM or TEM. The medium was changed at day 1 and day 2. Time-lapse imaging was then performed using a Lionheart™ FX Fluorescence Microscope (BioTek). Imaging was performed from D2 to D8 at 20 min-intervals, for a total of 500 times for each sample, and a movie was generated for each analyzed field. In order to calculate the conversion efficiency, we counted cell divisions of approximately 50 cells in each movie (n = 5) with TEM. For the colony formation assay, 2,000 PHCs were seeded on 6-well Matrigel-coated plates and stained with crystal violet (Sigma-Aldrich) at day 10. Whole-well scanning were performed using the Lionheart™ FX microscope.
Lionheart fx fluorescence microscope
Manufactured by Agilent Technologies
Sourced in United States
The Lionheart™ FX Fluorescence Microscope is a laboratory instrument designed for fluorescence imaging. It is capable of capturing high-quality fluorescence images and videos.
Automatically generated - may contain errors
Lab products found in correlation
Crystal violet, by Merck Group (1 mentions)
Apopxin green indicator, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Evos xl digital imaging system, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti β tubulin, by Merck Group (1 mentions)
3 protocols using lionheart fx fluorescence microscope
1
Quantifying PHC Conversion Efficiency
2
Apoptosis Induction and Detection
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Cells were subjected to starvation of glucose and/or amino acids or treated with phenformin and incubated during the indicated period. Cell extract was obtained using the lysis buffer for WB and analyzed for cleaved PARP and caspase 3 by WB. Additionally, cells were stained using pSIVA-IANBD specific to apoptotic cell membrane (Abcam, ab129817). Cell images were acquired using Lionheart FX fluorescence microscope (Bio-Tek, Winooski, VT) and analyzed using Gen5 program (version 3.10). Apoptotic and viable cells were also stained using apopxin green indicator and cytocalcein violet 450 (Abcam, ab176749), respectively, following the manufacturer’s protocol, and analyzed by NovoCyte Quanteon flow cytometry (Agilent Technologies, Santa Clara, CA) with 488 and 405 nm as the excitation lasers and 530/30 and 445/45 nm as the detection channels.
3
Immunofluorescence Analysis of Bothrops Venom
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Nuclear and cell morphology were analyzed by photographed images in bright field microscopy with a 20x objective (Life Technologies EVOS XL Digital Imaging System) and by immunofluorescent staining. BRL-3A cells were cultured on coverslips in 35 mm plates and treated with each Bothrops spp. venoms for 24 h, as described before. Cells were fixed with formaldehyde (3.7%) for 30 min and permeabilized with Triton X-100 (0.5%) for 30 min [29 (link)]. The samples were washed with PBSA, immunostained with primary monoclonal antibodies produced in mice, anti-α-tubulin (1:50) and anti-β-tubulin (1:50) (Sigma-Aldrich, MO, USA), and incubated overnight at room temperature in the dark. Corresponding secondary antibody anti-mouse IgG (H + L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) (1:50) (Cell Signaling Technology®, MA, USA), was incubated for 2 h. Actin microfilaments were labeled with phalloidin conjugated to Alexa Fluor® 555 (1:20) (Sigma-Aldrich, MO, USA) for 2 h. Nuclei were labeled with DAPI (1:100) (Sigma-Aldrich, MO, USA), and the coverslips were mounted with Vecta-Shield (Vector Laboratories, CA, USA). Analyses were performed on the LionHeart FX fluorescence microscope (Biotek, VT, USA).
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