Aug 07, 2023

Public workspaceFM1-43 endocytic uptake assay in HIPSC derived neurons

DOI
dx.doi.org/10.17504/protocols.io.3byl4qj52vo5/v1
  • 1Waisman Center, University of Wisconsin-Madison, Madison, WI
Daniel El Kodsi
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DOI: dx.doi.org/10.17504/protocols.io.3byl4qj52vo5/v1
Protocol Citation: Sakthi Kumar 2023. FM1-43 endocytic uptake assay in HIPSC derived neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4qj52vo5/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: August 07, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86065
Keywords: ASAPCRN, Endocytosis, FM1-43
Funders Acknowledgement:
MJFF
Grant ID: 000301
Abstract
FM1-43 is a lipophilic styryl dye, nontoxic to the cells, virtually nonfluorescent in aqueous media. The dye will become fluorescent once it binds to the plasma membrane. In neurons that are actively releasing neurotransmitters, these dyes become internalized within the recycled synaptic vesicles, and the nerve terminals become brightly stained. This shows an increase in the fluorescence intensity of FM1-43. When the cells undergo another stimulation (through KCL or electrical) the FM1-43 dye containing vesicles will be released through exocytosis. Thus, resulting in a decrease in the FM1-43 fluorescence intensity.
Reagents needed:
Reagents needed:
FM1-43 dye (Thermo fisher scientific, catalog no: T3163).
This dye is available in both live and fixable versions. Also, available in different colors like FM4-64
Mature neurons- hipsc derived Dopaminergic, Cortical or GABAergic neurons:
Mature neurons- hipsc derived Dopaminergic, Cortical or GABAergic neurons:
3w
3w
  1. mDA, CTX or GABAergic neuron progenitors were plated in 12mm coverslips, precoated with PDL+ Laminin.
  2. After Duration504:00:00 post plating, neurons were subjected to FM1-43 dye uptake. Note: We have not tried in early immature neurons; this experiment was also performed in more mature neurons like 40 days post plating.
  3. Some cell types may mature differentially. So, the user must validate the protocol based on their cell type and other experimental conditions.

3w
Required buffer:
Required buffer:
  1.     ACSF
  2.     ACSF- containing high KCL (50mM) for stimulation.
  3.     ACSF- containing no Calcium to prevent spontaneous release.

Normal ACSF composition:
Standard External (pH 7.4 NaOH)
 milliMolarGms/500ml
NaCl1404.090 gms
KCl40.150
CaCl220.150
MgCl220.204
TES(HEPES)10(10)1.146(1.191)
Glucose50.440
Spontaneous FM1-43 uptake:
Spontaneous FM1-43 uptake:
10m
10m
  1. Transfer the coverslips to normal ACSF.
  2. Add 10uM of FM1 43 dye to the cells and wait for Duration00:10:00 at TemperatureRoom temperature .
  3. Remove the dye and add ACSF without calcium and wash for 3 times. At this step, the user may want to add antagonists for AMPA/ NMDA or Na+ channel to inhibit spontaneous release.
  4. After 3 washes transfer the coverslips to the imaging chamber (for live imaging) or add PFA for fixation.


10m
PFA fixation:
PFA fixation:
25m
25m
  1.     After adding 4% PFA wait for Duration00:15:00 for fixation.
  2.     Then wash 3 times with PBS.
  3.     Add Hoechst and incubate for Duration00:10:00 .
  4.     Then wash 3 times with PBS and mount the coverslips on slides with Fluoromount G.
  5.     Image the same day or keep the slides at Temperature4 °C if imaging the next day.


25m
Stimulation induced FM1-43 dye uptake:
Stimulation induced FM1-43 dye uptake:
11m
11m
  1.     Transfer the coverslips to normal ACSF.
  2.     Add ACSF containing high KCL (50mM) for Duration00:01:00 to stimulate the cells.
  3.     Then Add 10um of FM1 43 dye to the cells and wait for Duration00:10:00 at TemperatureRoom temperature to measure the post stimulus induced endocytosis.
  4.     Remove the dye and add ACSF without calcium and wash for 3 times. At this step, the user may want to add antagonists for AMPA/ NMDA or Na+ channel to inhibit spontaneous release.
  5.     After 3 washes transfer the coverslips to the imaging chamber (for live imaging) or add PFA for fixation.
  6.     If fixing the cells, follow the same steps as mentioned above in PFA fixation.



11m
Quantification:
Quantification:
Use ImageJ based program to quantify FM1-43 fluorescence intensity.