FeRIC-based magnetogenetics: evaluation of methods and protocols in in vitro models

TL;DR: Three in vitro protocols were compared and several experimental factors were examined that either enhance or abolish the RF control of ferritin-tagged TRPV to help establish reproducible magnetogenetic experimental protocols.

Abstract: FeRIC (Ferritin iron Redistribution to Ion Channels) is a magnetogenetic technique that uses radiofrequency (RF) waves to activate the transient receptor potential channels, such as TRPV1 and TRPV4, coupled to cellular ferritins. In cells expressing ferritin-tagged TRPV, RF stimulation increases the cytosolic Ca2+ levels via a biochemical pathway. The interaction between RF and ferritin increases the free cytosolic iron level that in turn, triggers chemical reactions producing reactive oxygen species and oxidized lipids that activate the ferritin-tagged TRPV. In this pathway, it is expected that experimental factors that disturb the ferritin expression, the ferritin iron load, the TRPV functional expression, or the cellular redox state will impact the RF efficacy to activate ferritin-tagged TRPV. Here, three in vitro protocols were compared for using FeRIC to remotely activate ferritin-tagged TRPV. Further, several experimental factors were examined that either enhance or abolish the RF control of ferritin-tagged TRPV. The findings may help establish reproducible magnetogenetic experimental protocols.

Summary

Introduction

• Magnetogenetics is a terminology loosely used to describe a group of techniques that apply magnetic fields to control cell activity via interactions with certain proteins.
• Other theoretical estimations, assuming the ferritin is at the maximum iron load (~ 4500 iron atoms) and has specific magnetic properties , have proposed potential mechanisms, such as the magnetocaloric effect, that might contribute to magnetic heating and subsequent activation of TRPV (Barbic, 2019; Duret et al., 2019) .
• There is no conclusive experimental evidence supporting those mechanistic proposals.
• This biochemical pathway and the role of ROS in the RFinduced activation of ferritin-tagged TRPV have been recently corroborated (Brier et al., 2020) .

I.

• FeRIC First, the authors estimated the distribution of the electric (E) and magnetic (B) fields applied to the cells to rule out the potential contribution of the E field to RF-induced Ca 2+ responses.
• To estimate the distribution of the E and B fields, the authors computed them using the finite-difference timedomain (FDTD) method implemented by the openEMS project (Liebig et al., 2013) .
• In contrast, the E field amplitude drastically decreases in the center of the coil/dish.
• 2004; Zmeykina et al., 2020) , they should be negligible and should not produce significant effects on the membrane ion channels.

II. RF-induced Ca

• 2+ responses in TRPV4 FeRIC -expressing N2a cells decreases with longer periods of TRPV4 FeRIC transient expression.
• In the literature, three main groups have reported successful magnetic control of ferritintagged TRPV channels expressed in diverse cultured cells (Brier et al., 2020; Hernández-Morales et al., 2020; Hutson et al., 2017; Stanley et al., 2016; Wheeler et al., 2016) .
• The in vitro protocols used among those studies vary in time delays between seeding, transfection, and Ca 2+ imaging.
• Timing protocol 1 (cells imaged 24-h post-transfection) is that the authors previously reported using FeRIC channels (Hernández-Morales et al., 2020; Hutson et al., 2017) .
• For all protocols, RF stimulation did not change the cytosolic Ca 2+ levels in N2a cells expressing only GCaMP6 and the functional expression of TRPV4 FeRIC was corroborated with GSK101 at 1 µM.

with RF fields

• Based on the observations described in this report, the authors have the following recommendations for FeRIC-based magnetogenetic techniques for RF-induced activation of cells in in vitro systems.
• For a detailed methodological description, please refer to the STAR Methods section.
• Cultured cells are allowed to transiently express the ferritin-tagged TRPV up to 24-h post-transfection.
• The authors observed that HTF at high concentration (above 1000 µg/mL) resulted in an appearance of intracellular vacuoles.
• This can be easily achieved by supplementing the culture medium with the Ca 2+ chelator EGTA.

Discussion

• Because the main components of ferritin-based magnetogenetics, including ferritin and TRPV, are subjected to a diversity of cellular regulatory mechanisms, it is crucial to unify the experimental protocols to obtain reproducible results.
• Here the authors report that RF induces reproducible Ca 2+ responses in cells expressing TRPV4 FeRIC at temperatures that are physiologically relevant.
• Increasing the cellular iron import may increase the ferritin iron load (Brier et al., 2020) and consequently enhances the RF-induced activation of TRPV4 FeRIC .
• Notably, the functional expression of TRPV is regulated by activity-dependent mechanisms, resulting in dynamic trafficking between the cell membrane and the cytosolic vesicle pool.
• The authors findings pointed out that ferritin-based magnetogenetics are sensitive to diverse experimental factors that may disturb the functional expression and function of ferritin and/or TRPV.

Limitations of the study

• This study is limited to a single ferritin-based magnetogenetic technique called FeRIC.
• Further studies are needed to corroborate their findings in other magnetogenetic approaches that use, for example, cells stably expressing the ferritin-tagged TRPV or chimeric ferritins.

Data and code availability

• The Magnetic/electric field simulation code used during this study is available at GitHub: https://github.com/LiuCLab/FeRIC/blob/master/FeRIC_FDTD_simulation.m. .
• The dataset identifiers and accession numbers are in the key resources table.
• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Cell lines

• N2a cells were obtained from the UCB Cell Culture Facility (University of California Berkeley).
• Cell identity and negative Mycoplasma contamination were verified by the UCB Cell Culture Facility.
• Cells were maintained in Dulbeccos's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, hyclone) and 100 units/mL penicillin, and 100 mg/mL streptomycin at 37°C and 5% CO2.
• For described experiments, cell lines were employed between the passages 5 to 20.

RF coils

• The coils were connected in series with tuning capacitors forming an LC circuit, and were tuned to a resonance frequency of about 180 MHz or 465 kHz.
• Using the Beehive probe and the Rigol spectrum analyzer, the magnetic field strength was measured to be about 1.6 µT for 180 MHz or 31 µT for 465 kHz at a location about 3 mm above the cell culture dishes.
• Next, with reflected illumination, the fluorescence signals from mCherry and GCaMP6 were corroborated and the field of interest was selected.
• Next, cells were washed three times with HBSS and rested for 30 min at room temperature in darkness.
• Transfection mix had the following composition per each 35-mm dish: 300 µL OptiMEM, 4 µL Lipofectamine LTX, 3 µL PLUS reagent, 0.7 µg TRPV4 FeRIC or TRPV4 WT DNA.

Quantification and statistical analysis

• The distribution of the electric (E) and magnetic (B) fields applied to the cells were simulated using the finite-difference time-domain (FDTD) method implemented by the openEMS project (Liebig et al., 2013) (https://openems.de/start/).
• The simulations were done considering the 5 cm-diameter RF coil containing the 3.5 cm-diameter dish half-filled with imaging saline solution (dish height: 1 cm, saline solution height: 0.5 cm).
• The dielectric constant (80) and conductivity (1.5 S/m) for the imaging saline solution was obtained from the literature (Davis et al., 2020) .
• The number of total cells (Hoechst 33342 stained cell nucleus) and the number of Yo Pro 1 positive cells in a field of view was computed in a cell-based analysis with a customized MATLAB (Release 2018b, MathWorks Inc., Natick, Massachusetts) code.

Quantification and Statistical Analysis

• All experiments were repeated a minimum of three times.
• Differences in continuous data sets were analyzed using Microcal OriginPro 2020 software .
• (J) Images of N2a cells expressing TRPV4 FeRIC (mCherry+) without stimulation or following GSK101 (50 nM) application for 20 min in the absence or the presence of GSK219.
• For each experimental condition, listed here is the averaged GCaMP6 or Fluo-4 area under the curve (AUC) ± SEM, the fraction of cells responsive to RF, the number of separate experiments (N), and the number of analyzed cells (n).

Full text

1
EVALUATING METHODS AND PROTOCOLS OF FERRITIN-BASED
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MAGNETOGENETICS
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4
Miriam Hernández-Morales
1,2
, Victor Han
1,2
, Richard H Kramer
3
, Chunlei Liu
1,2
*
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6
1
Department of Electrical Engineering and Computer Sciences, University of California,
7
Berkeley, CA 94720, USA
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2
Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720, USA
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3
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA
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94720, USA
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Figures: 4
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Supplemental figures: 4
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Supplemental tables: 1
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Lead contact:
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*Chunlei Liu, PhD
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505 Cory Hall MC# 1770
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University of California, Berkeley, CA 94720, USA
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Email: chunlei.liu@berkeley.edu
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Tel: (510)664 7596
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted September 7, 2021. ; https://doi.org/10.1101/2020.12.10.419911doi: bioRxiv preprint

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Summary
1
FeRIC (Ferritin iron Redistribution to Ion Channels) is a magnetogenetic technique that uses
2
radio frequency (RF) alternating magnetic fields to activate the transient receptor potential
3
channels, TRPV1 and TRPV4, coupled to cellular ferritins. In cells expressing ferritin-tagged
4
TRPV, RF stimulation increases the cytosolic Ca
2+
levels via a biochemical pathway. The
5
interaction between RF and ferritin increases the free cytosolic iron levels that in turn, trigger
6
chemical reactions producing reactive oxygen species and oxidized lipids that activate the
7
ferritin-tagged TRPV. In this pathway, it is expected that experimental factors that disturb the
8
ferritin expression, the ferritin iron load, the TRPV functional expression, or the cellular redox
9
state will impact the efficiency of RF in activating ferritin-tagged TRPV. Here, we examined
10
several experimental factors that either enhance or abolish the RF control of ferritin-tagged
11
TRPV. The findings may help optimize and establish reproducible magnetogenetic protocols.
12
13
Subject areas
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Magnetogenetics, neuromodulation, radiofrequency magnetic fields
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted September 7, 2021. ; https://doi.org/10.1101/2020.12.10.419911doi: bioRxiv preprint

3
1
Introduction
2
Magnetogenetics is a terminology loosely used to describe a group of techniques that
3
apply magnetic fields to control cell activity via interactions with certain proteins. This group of
4
techniques uses a diverse range of magnetic field conditions to target various choices of
5
membrane proteins. These differences have contributed to the confusion and debates regarding
6
the biophysical mechanisms and experimental reproducibility. In one class of magnetogenetic
7
techniques, static, low frequency or radio frequency (RF) alternating magnetic fields have been
8
applied to activate transient receptor potential vanilloid channels (TRPV) coupled to ferritin (Brier
9
et al., 2020; Duret et al., 2019; Hernández-Morales et al., 2020; Hutson et al., 2017; Stanley et
10
al., 2016). The biophysical mechanisms responsible for magnetic activation of the channels are
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still being worked out. It was first proposed that the interaction between static or RF magnetic
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fields and ferritin produces heat or mechanical stimuli that directly activate TRPV1 and TRPV4,
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which are intrinsically temperature- and mechanically-sensitive (Stanley et al., 2016; Wheeler et
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al., 2016). However, no change in temperature could be detected at the surface of ferritin upon
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RF exposure (Davis et al., 2020). Likewise, a theoretical calculation estimates that the
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temperature change produced by interaction of magnetic fields with ferritin is several orders of
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magnitude lower than that required to activate TRPV (Meister, 2016). Other theoretical
18
estimations, assuming the ferritin is at the maximum iron load (~ 4500 iron atoms) and has
19
specific magnetic properties (superparamagnetic), have proposed potential mechanisms, such
20
as the magnetocaloric effect, that might contribute to magnetic heating and subsequent
21
activation of TRPV (Barbic, 2019; Duret et al., 2019). However, there is no conclusive
22
experimental evidence supporting those mechanistic proposals. Recently, we proposed an
23
indirect biochemical mechanism that allows RF to activate ferritin-tagged TRPV. Specifically, by
24
triggering dissociation of iron from the ferritin, RF catalyzes the generation of reactive oxygen
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species (ROS), short-chain fatty acids, and oxidized lipids, which are activators of TRPV
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(Hernández-Morales et al., 2020). This biochemical pathway and the role of ROS in the RF-
27
induced activation of ferritin-tagged TRPV have been recently corroborated (Brier et al., 2020).
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Besides the uncertainties about the underlying mechanisms, the efficiency of a specific
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magnetogenetic technique that uses a ferritin-fused TRPV4, named Magneto2.0 (Wheeler et al.,
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2016) has been questioned. Three independent groups reported the failure to activate neurons
31
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted September 7, 2021. ; https://doi.org/10.1101/2020.12.10.419911doi: bioRxiv preprint

4
expressing Magneto2.0 upon stimulation with static magnetic fields (Kole et al., 2019; Wang et
1
al., 2019; Xu et al., 2019). It has been proposed that diverse experimental factors are responsible
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for those discrepancies such as the magnetic stimuli (static versus low frequency magnetic
3
fields), the viral vectors used to deliver Magneto2.0 (pAAV versus Semliki Forest virus, Sindbis
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virus, and lentivirus), and the transduction period to achieve functional expression of Magneto2.0
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(Wheeler et al., 2020). However, there is no experimental evidence reported to support the
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hypothesis that those specific experimental factors are responsible for conflicting results using
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Magneto2.0.
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The lack of a unified experimental protocol has compounded the many unresolved issues.
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For example, ferritin-based magnetogenetic approaches use diverse magnetic stimuli (static,
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low frequency, kHz RF, or MHz RF magnetic fields) and have tagged TRPV with both
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endogenous or chimeric ferritin (Brier et al., 2020; Duret et al., 2019; Hernández-Morales et al.,
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2020; Hutson et al., 2017; Stanley et al., 2016; Wheeler et al., 2016). Other factors that may
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contribute to the reported inconsistencies are the function and expression of both ferritin and
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TRPV. Ideally, ferritins should be at the maximum iron load to transduce magnetic fields
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proficiently. Nevertheless, it is unknown if the chimeric ferritins store iron at the same level as
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endogenous ferritins. Furthermore, the iron load of ferritins is highly variable from almost empty
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up to maximum load (~4500 iron atoms) (Jian et al., 2016). Regarding TRPV, those channels
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should be functionally expressed at the cell membrane. However, their expression and sensitivity
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to diverse stimuli are subjected to cellular regulatory mechanisms. Several TRPV channels are
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constitutively active, and cells prevent the associated cytotoxic effect by downregulating the
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TRPV density at the plasma membrane (Ferrandiz-Huertas et al., 2014; Montell, 2004; Planells-
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Cases and Ferrer-Montiel, 2007).
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Given the many potential variables described above, we reasoned that experimental
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factors that disturb the cellular iron homeostasis and the ferritin and TRPV channel expression
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may impact the magnetic control of ferritin-tagged TRPV. Using a single magnetogenetic
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approach, FeRIC technology, we examined the influence of diverse experimental variables on
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the RF control of cytosolic Ca
2+
levels in cells expressing ferritin-tagged TRPV4. Interestingly,
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we found that while some experimental variables abolished magnetic control of ferritin-tagged
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TRPV4, others enhanced it. The observations reported here may contribute to the
30
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted September 7, 2021. ; https://doi.org/10.1101/2020.12.10.419911doi: bioRxiv preprint

5
standardization and optimization of current and future magnetogenetic techniques to achieve
1
better reproducibility.
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3
Results
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I. FeRIC technology allows the magnetic control of cytosolic Ca
2+
levels
5
Here we examined the influence of different experimental factors on the Ca
2+
responses induced
6
by RF fields in cultured cells expressing the ferritin-tagged channel TRPV4
FeRIC
. RF fields at 180
7
MHz and 1.6 µT were generated with solenoid coils enclosing a cell culture dish (Figure S1A,
8
B). The cytosolic Ca
2+
levels were monitored in Neuro2a (N2a) cells expressing GCaMP6 plus
9
TRPV4
FeRIC
. All experiments were performed at room temperature (22ºC) except for those
10
testing TRPV4
FeRIC
responsiveness at 32ºC and 37ºC. The cells expressing TRPV4
FeRIC
were
11
identified using the mCherry reporter. Data were quantified as the change in GCaMP6
12
fluorescence divided by baseline fluorescence (DF/F0), the GCaMP6 area under the curve
13
(AUC), and the fraction of cells responsive to RF (RF responsiveness). A cell was considered
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RF responsive when the GCaMP6 DF/F0 increased 10 times over the standard deviation of its
15
baseline fluorescence (see STAR methods).
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First, we estimated the distribution of the electric (E) and magnetic (B) fields applied to the
17
cells to rule out the potential contribution of the E field to RF-induced Ca
2+
responses. To
18
estimate the distribution of the E and B fields, we computed them using the finite-difference time-
19
domain (FDTD) method implemented by the openEMS project (Liebig et al., 2013). The
20
simulation setup included the 5 cm-diameter RF coil containing the 3.5 cm-diameter dish half-
21
filled with imaging saline solution (dish height: 1 cm, saline solution height: 0.5 cm) (Figure S1C).
22
The estimated E field corresponding to a 180 MHz and 1.6 µT magnetic field, at the center of
23
the culture dish, was about 5.5 V/m. The magnitude distributions in two dimensions, for both
24
transverse and longitudinal cross-sections, of the B and E fields are shown in Figure 1A. As
25
expected, the strength of the B field remains relatively large at the center of the coil/dish. In
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contrast, the E field amplitude drastically decreases in the center of the coil/dish. Although the
27
estimated electric field values for the RF at 180 MHz and 1.6 µT are similar in amplitude to those
28
needed for transcranial magnetic stimulation (TMS) (Fox et al., 2004; Zmeykina et al., 2020),
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they should be negligible and should not produce significant effects on the membrane ion
30
channels. Firstly, the induced potential difference between the two opposite sides of a cell
31
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted September 7, 2021. ; https://doi.org/10.1101/2020.12.10.419911doi: bioRxiv preprint

Frequently asked questions

Q1. How long did the cells rest before applying the RF stimulus?

15 Because increasing the temperature from 22ºC to 32-37ºC activates TRPV4FeRIC, cells were 16 rested for ~20 min before applying the RF stimulus. 

Q2. How can the authors achieve reproducible results with RF?

Because the main components of ferritin-based magnetogenetics, including ferritin and 8 TRPV, are subjected to a diversity of cellular regulatory mechanisms, it is crucial to unify the 9 experimental protocols to obtain reproducible results. 

Q3. What was the magnetic field strength measured using the TekBox probe?

Using the Beehive 12 probe and the Rigol spectrum analyzer, the magnetic field strength was measured to be about 13 1.6 µT for 180 MHz or 31 µT for 465 kHz at a location about 3 mm above the cell culture dishes. 

Q4. Why were the GCaMP6 fluorescence measurements discarded?

For analysis, the 27GCaMP6 fluorescence measurements corresponding to the first 5 frames were discarded 28 because of the appearance of an inconsistent artifact. 

Q5. How many cDNAs were used to generate the TRPV4WT construct?

The full-length wild-type TRPV4 was 18 subcloned into the PLVX-IRES-mCherry vector to generate TRPV4WT (Clontech, Catalog No. 19 631237). 

Q6. How is the cytosolic Ca2+ levels in ferritin-tagged cells?

67 1. Using their RF system, RF fields at high MHz frequencies are efficient in increasing the 8 cytosolic Ca2+ levels in cells expressing ferritin-tagged TRPV. 

Q7. How can the authors reduce the levels of ferritin-tagged TRPV?

For those cases where it is not possible to allow for short time periods of ferritin-tagged 1 TRPV expression or to supplement the culture medium with HTF or apoTf, the functional 2 downregulation of those channels can be decreased by lowering the extracellular Ca2+ 3 levels. 

Q8. How did the HTF treatment affect the GCaMP6 AUC?

in TRPV4FeRIC-23expressing N2a cells imaged 72-h after transfection, 500 µg/mL HTF treatment produced about 24a 10-fold increase in the RF-induced increase of GCaMP6 AUC relative to non-treated cells 25 (Figure 2D, E; Table S1). 

Q9. What is the effect of Fura-2 on GCaMP6?

It has been reported that some Ca2+ dyes, such as Fura-2, may interfere with intracellular 7 Ca2+ signaling (Alonso, 2003; Smith et al., 2018). 

Q10. What is the role of temperature in the downregulation of TRPV?

their 10 observations indicate that abolishing the temperature sensitivity of TRPV or lowering the 11 extracellular Ca2+ levels prevent the functional downregulation of ferritin-tagged TRPV. 

Q11. How many independent groups reported the failure to activate Magneto2.0 upon stimulation with static magnetic fields?

Three independent groups reported the failure to activate neurons 314expressing Magneto2.0 upon stimulation with static magnetic fields (Kole et al., 2019; Wang et 1 al., 2019; Xu et al., 2019). 

Q12. How many seconds are the kinetics of the RF-induced Ca2+ responses?

the authors acknowledge that 13 the activation and decay kinetics of the RF-induced Ca2+ responses with FeRIC technology are 14 in the hundreds of second scale.