For the soft agar transformation assay, 2.5 × 104 cells were seeded in 1 ml of regular medium with 0.5% low melting point agarose and overlaid on 1 ml of medium with 1% agarose in each well of a six-well plate. After 3 weeks, colonies larger than 100 μm in diameter were counted. The wound healing assay was performed using a culture-insert (ibidi GmbH, Germany) according to manufacturer's instruction. The culture-insert had two cell culture reservoirs, which were separated by a 500 μm-thick wall. Same numbers of cells were seeded in the culture-insert. After 24 h, the culture-insert was removed, which left a cell-free “wound” of around 500 μm in width. The wound closure was observed by a time lapse microscopy (Zeiss, Germany) and images were obtained at 1 h interval for 24 h. The area of wound was analyzed using the ImageJ software program.
Time lapse microscopy
Manufactured by Zeiss
Sourced in Germany
Time-lapse microscopy is a technique that captures a series of images over an extended period, allowing for the observation of dynamic processes within a sample. This equipment enables the visualization of changes, growth, or movement occurring at the cellular or subcellular level.
Automatically generated - may contain errors
Lab products found in correlation
Culture insert, by Ibidi (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Axio observer z1, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Calibre Scientific (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Vincristine, by Merck Group (1 mentions)
Lidocaine, by Merck Group (1 mentions)
Axio observer z1 system, by Zeiss (1 mentions)
5 protocols using time lapse microscopy
1
Soft Agar and Wound Healing Assays
2
Visualization of Keratinocyte Wound Healing
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One million HaCaT keratinocytes were seeded onto 60 mm plastic culture dishes and incubated overnight to permit cell adhesion. Scratches were performed using a 10 μL pipette tip. Gap closure was followed by time lapse microscopy (Zeiss, Germany) and three gap distances per samples were evaluated at different time points using Axio Vision software (Zeiss). For immunofluorescence, HaCaT keratinocytes were grown on glass coverslips for 24 h prior to fixation (4% paraformaldehyde; Sigma) and permeabilization (PBS with 0.1% Triton X-100; Sigma). Samples were subsequently incubated with primary antibodies (1 : 500; cell signaling technologies, USA) targeted at zona occludens protein 1 (ZO-1) overnight at 4°C. Cells were washed twice and incubated with appropriate Alexa Fluor 488-conjugated secondary antibodies (1 : 700; life technologies) for 1 h. Coverslips were washed and mounted onto glass microscope slides using mounting medium containing DAPI (VectaShield; Biozol, Germany) prior to analysis using an Axio Observer Z.1 (Zeiss).
3
Neuronal Transport Assay with PAD2 Overexpression
4
Temporal Dynamics of Hepatocyte Cytokinesis
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The cytokinetic structures of hepatocytes were monitored in vitro by time-lapse microscopy (Carl Zeiss). Primary cultured hepatocytes isolated from the livers of 3-week-old mice were used since the proliferation ability of hepatocytes at this age is most vigorous and over 90% of hepatocytes are diploid and tetraploid (Fig. 1h,i ). 24 h after seeding, images of hepatocytes were recorded live with a high resolution wide field inverted microscope Axio Observer Z1 system (Carl Zeiss). Cellular structures were visualized by phase contrast system with A-Plan 10 × / 0.25 dry objective lens and automatic exposure time. Hepatocytes were incubated in a chamber maintained at 37 °C and 5% CO2 (CO2/Temp Module S, Carl Zeiss). Images were taken at 10 min intervals for approximately 120 h by AxioCam MRm CCD camera. The optimal focal plane was set at beginning of each image session and adjusted by Definite Focus system with 10-s intervals throughout the image recording. The recorded images were analyzed via ImageJ software.
5
PAH-induced Cell Migration Assay
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MCF7, C33A and SiHa cells were treated with 1 μM of PAH for three days prior to analysis by wound healing assay. Then 3 × 105 MCF7 cells, 4 × 105 C33A cells and 2 × 105 SiHa cells were seeded into a 24-well plate so that cells would nearly reach confluency the next day. A wound was then introduced to the confluent cell monolayer by scratching with a 10-μL pipette tip. Cells were washed twice with phosphate-buffered saline (PBS) and monitored by time-lapse microscopy (Zeiss, Jena, Germany) for 48 h in DMEM supplemented with 2% FBS and 1 μM of PAH. Images were captured every hour. Cell migration index was calculated as the cell migrated area (T0 - T48) of experimental group divided by the control group in each cell line.
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