Dec 22, 2024

Public workspaceCalcium imaging in human dorsal root ganglia neurons

DOI
dx.doi.org/10.17504/protocols.io.n2bvj9rznlk5/v1
  • 1University of Texas at Dallas
Keerthana Natarajan
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DOI: dx.doi.org/10.17504/protocols.io.n2bvj9rznlk5/v1
Protocol Citation: Keerthana Natarajan, Úrzula Franco-Enzástiga, Felipe Espinosa, Theodore Price 2024. Calcium imaging in human dorsal root ganglia neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj9rznlk5/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: November 03, 2024
Last Modified: December 22, 2024
Protocol Integer ID: 111463
Keywords: Calcium Imaging, Fluo-4 AM, Human Dorsal Root Ganglia Neurons, Type I Interferon
Funders Acknowledgements:
National Institute of Health
Grant ID: NS130608
National Institute of Health
Grant ID: NS065926
Abstract
Recording capsaicin and high K+ induced calcium dynamics using Fluo-4 AM dye in cultured human dorsal root ganglia neurons treated with human interferon alpha (hIFN-α) or human interferon beta (hIFN-β).
Guidelines
All steps described in the protocol must be performed while wearing appropriate PPE, including a lab coat and gloves. Human neuronal cultures should be handled with proper safety precautions while handling and disposing.
Materials
Solutions:

Recording solution (pH 7.4; 300-310 mOsm):
  • 130 mM NaCl
  • 4.2 mM KCl
  • 1.1 mM CaCl2
  • 1 mM MgSO4
  • 0.45 mM NaH2PO4.H2O
  • 0.5 mM NaH2PO4.7H2O
  • 20 mM Glucose
  • 10 mM HEPES

High K+ solution (pH 7.4; 300-310 mOsm):
  • 84.2 mM NaCl
  • 50 mM KCl
  • 1.1 mM CaCl2
  • 1 mM MgSO4
  • 0.45 mM NaH2PO4.H2O
  • 0.5 mM NaH2PO4.7H2O
  • 20 mM Glucose
  • 10 mM HEPES

Materials and equipment:

  • Fluo-4 AM - Invitrogen (Cat no. F14201)
  • Pluronic F-127 - Invitrogen (Cat. no. P3000MP)
  • HBSS - Thermofisher Scientific (Cat. no. 14170161)
  • Recombinant human interferon alpha A protein - Sigma Aldrich (Cat. no. IF007)
  • Recombinant human interferon beta 1a protein - Sigma Aldrich (Cat. no. IF014)
  • Capsaicin - Sigma Aldrich (Cat. no. M2028)
  • Bath imaging chamber - Warner Instruments (Cat. no. RC-25)
  • Forceps - Fine Science Tools (Cat. no. 11252-00)
  • Elve Flow MUX Distribution 12-way Bidirectional Valve perfusion system
  • Olympus IX83 inverted microscope
  • Vapor Pressure Osmometer - Vapro (Cat. no. 5600)
  • Pipettes and corresponding pipette tips

Software/tools:

  • MetaFluor for Olympus imaging software (version 7.10.5.476)
  • Elveflow Smart Interface (version 3.07.03)
Solutions and dye preparation
Solutions and dye preparation
Prepare the recording solution with 130 mM NaCl, 4.2 mM KCl, 1.1 mM CaCl2, 1 mM MgSO4, 0.45 mM NaH2PO4.H2O, 0.5 mM NaH2PO4.7H2O, 20 mM glucose, and 10 mM HEPES. Using a vapor pressure osmometer, adjust the osmolarity to 300-310 mOsm. Adjust the pH to 7.4 using N-methyl-D-glucamine.
Note
The recording solution can be prepared and stored at 4 ℃. Equilibrate the recording solution to RT before imaging.

Prepare high K+ solution by adjusting KCl to 50 mM and NaCl to 84.2 mM in the recording solution, osmolarity 300-310 mOsm. Adjust the pH to 7.4 using N-methyl-D-glucamine.
Note
High K+ can be prepared and stored at 4 ℃. Equilibrate high K+ solution to RT before imaging.

Prepare 1 mL of 10 μM working solution of Fluo-4 AM with 0.04% Pluronic F-127 surfactant in HBSS. Store it away from light.
Treatment of hDRG neurons
Treatment of hDRG neurons
Treat cultured DIV 1-4 hDRG neurons with 500 U/mL hIFN-α2 or hIFN-β 1a or their corresponding vehicles (0.002% BSA in PBS or 50 mM NaOAc, pH 5.5, containing 0.1% BSA, respectively)Duration02:30:00 .
Note
Preparation of hDRG cultures followed previously described methods (see references).

Disclaimer: The duration of treatment depends on the experimental designs and cytokines used, and should be optimized accordingly.

2h 30m
Incubation
Setting up calcium imaging
Setting up calcium imaging
Attach the recording solution, 200 nM capsaicin dissolved in the recording solution, and the high K+ solution to the Elve Flow MUX Distribution 12-way Bidirectional Valve system for perfusion and set the flow rate to 1 mL/min.
Note
Disclaimer: The concentration of capsaicin can vary between experimental designs and hence, should be optimized accordingly.

Using a brush, coat the bottom of a bath imaging chamber with petroleum jelly.


Wash cells once with growth media.
Incubate cells at 37 °C with 10 μM Fluo-4 AMDuration00:30:00 .
30m
Remove the dye and wash the cells with 1 mL of HBSS.
Retrieve the coverslip with cultured hDRG neurons from the plate well with forceps and mount it to the imaging chamber coated with petroleum jelly to form a tight seal.


Note
Gently pipette ~50 μL of recording solution onto the cells to prevent them from drying out.

Place the imaging chamber on an Olympus IX83 inverted microscope at 10x magnification.


Begin perfusion of recording solution at a flow rate of 1 mL/min achieved by 100-120 kPa of nitrogenDuration00:05:00 .
Flow rate adjustment and channels on Elveflow Smart Interface (ESI) for the Elve Flow MUX Distribution 12-way Bidirectional Valve perfusion system.


5m
Capturing intracellular calcium changes in hDRG neurons
Capturing intracellular calcium changes in hDRG neurons
Open MetaFluor for Olympus imaging software. Choose the field of view to be imaged and bring it to focus.
Focusing field of view for imaging on MetaFluor.

Imaging
Acquire an image and select all neurons of interest by drawing a circular ROI around them. Keep in mind that at least one ROI in the background should be drawn to account for background fluorescence.

Regions tool on MetaFluor.

Circular ROI around hDRG neurons loaded with Fluo4-AM to read calcium dynamics.

Imaging
Select the background ROI drawn for the background fluorescence to be accounted for as a reference for subtraction.
Check "Log Data" to open an active spreadsheet that logs the fluorescence readout of each frame recorded.


Acquisition and data logging on MetaFluor.

Start acquiring images and recording baseline fluorescence indicative of baseline intracellular calcium levelsDuration00:10:00 .
10m
Imaging
After recording the baseline, switch to perfusion with 200 nM capsaicin solution to record capsaicin-induced intracellular calcium changes in neuronsDuration00:02:00 .
2m
Wash out capsaicin by perfusing the recording solutionDuration00:03:00 .
3m
Finally, perfuse 50 mM high K+ solutionDuration00:02:00 .
Graph representing varying intracellular calcium levels in hDRG neurons induced by capsaicin and high K+ as fluorescence intensity over time.

2m
Stop the solution perfusion and acquisition of images. Export the spreadsheet for further data analysis.
Note
Analysis of peak fold-change in fluorescence, latency to peak, and other metrics can be performed using Python, R, MATLAB, or other open-source or commercially available packages.

Analyze
Computational step
Protocol references
Yousuf MS, et al. (2023) Highly specific σ2R/TMEM97 ligand FEM-1689 alleviates neuropathic pain and inhibits the integrated stress response. Proc Natl Acad Sci USA 120(52):e2306090120.

David ET, et al. (2024) Ephrin-B2 promotes nociceptive plasticity and hyperalgesic priming through EphB2-MNK-eIF4E signaling in both mice and humans. Pharmacol Res. 206:107284.
Acknowledgements
We thank Anna Cervantes, Geoffrey Funk, and Peter Horton at the Southwest Transplant Alliance. We are grateful to the organ donors and their families for their donations.