Jul 03, 2023

Public workspaceActin flow

DOI
dx.doi.org/10.17504/protocols.io.5jyl8pmb8g2w/v1
  • 1University of Glasgow
Juan.Gonzalez
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DOI: dx.doi.org/10.17504/protocols.io.5jyl8pmb8g2w/v1
Protocol Citation: Juan.Gonzalez 2023. Actin flow . protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8pmb8g2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 01, 2023
Last Modified: July 03, 2023
Protocol Integer ID: 84352
Abstract
Actin flow
Experimental procedure
Experimental procedure
Transfect the cells with the LifeAct-GFP plasmid following the protocol "Cells electroporation for cell transfection with NEON system"
The next day, trypsinize the cells as usual

Seed the cells on glass (i.e. gels on glass bottom petri dishes or the glass itself)


Note
The cell density depends on the cell type used. i.e. for C2C12 myoblasts, we seed 10000 cells/cm2

After Duration03:00:00 , image the cells with a confocal microscope. Use the 40x objective. If the CO2 cannot be controlled, use CO2-independent medium. The channel would be eGFP.

3h
Focus the cell in a focal plane where the actin cytoskeleton can be seen


We perform a time series of 120 images every Duration00:00:02 (Duration00:04:00 in total per cell)
4m 2s
Data analysis
Data analysis
Open the image sequence with Fiji software

Expected result

Video


Analyze, Set measurements, select “Bounding rectangle” and “Min & Max gray value”
Check where the cell cytoskeleton moves
Draw a line with the wand tool crossing the movement
Analyze, Multi kimograph, multi kymograph, linewidth: 1
A black & white gradient pops up. White lines indicate cell movement. The higher angle indicates more movement
Draw a line with the wand tool in the white line seen in the kymograph


Expected result
Data analysis from kymograph with Fiji software



Press “M” and copy & paste the measurements in an Excel file
In the Excel file, split the width by the length. This new value means flow in "pixels per frame"
Divide the pixels/frame value obtained by 2. This new value means flow in "pixels per second"
Split the "pixels/s" value by the "pixels/micron" ratio of the objective used (i.e. 3.2055 in the LSM900 confocal microscope) and multiply by 1000. This final value is the actin flow in nm/s