Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor (2024)

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Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor (1)

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STAR Protoc. 2024 Jun 21; 5(2): 103130.

Published online 2024 Jun 12. doi:10.1016/j.xpro.2024.103130

PMCID: PMC11225890

PMID: 38870018

Samuel E. Holtzen,1,2 Ananya Raksh*t,1 and Amy E. Palmer1,3,4,

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Associated Data

Supplementary Materials
Data Availability Statement


Zinc (Zn2+) plays roles in structure, catalysis, and signaling. The majority of cellular Zn2+ is bound by proteins, but a fraction of total Zn2+ exists in a labile form. Here, we present a protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded Förster resonance energy transfer (FRET) sensor. We describe steps for producing buffered Zn2+ solutions for performing an imaging-based calibration and analyzing the imaging data generated to determine labile Zn2+ concentration in single cells.

For complete details on the use and execution of this protocol, please refer to Raksh*t and Holtzen etal.1

Subject areas: Single Cell, Cell-based Assays, Microscopy, Molecular/Chemical Probes, Chemistry

Graphical abstract

Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor (2)

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  • Generate a highly precise buffered Zn2+ solution using a two-ion buffer system

  • Calibrate a Zn2+-specific FRET sensor via fluorescence microscopy

  • Analyze microscopy images and calculate cytosolic-free Zn2+

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Zinc (Zn2+) plays roles in structure, catalysis, and signaling. The majority of cellular Zn2+ is bound by proteins, but a fraction of total Zn2+ exists in a labile form. Here, we present a protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded Förster resonance energy transfer (FRET) sensor. We describe steps for producing buffered Zn2+ solutions for performing an imaging-based calibration and analyzing the imaging data generated to determine labile Zn2+ concentration in single cells.

Before you begin

The metal ion zinc (Zn2+) is an essential micronutrient. It binds thousands of proteins and plays roles in catalysis and protein structure.2,3 Although there are hundreds of micromolar total Zn2+ in a typical mammalian cell, most of this is bound, buffered, or otherwise sequestered.4 The labile Zn2+ levels in cells vary by cell type but are typically on the order of hundreds of picomolar.1,5,6,7 In response to extracellular and intracellular signals, cells can increase or decrease labile Zn2+ by releasing it from intracellular stores such as buffering proteins and organelles.

In recent years, fluorescent biosensors based on Förster resonance energy transfer (FRET) have become prevalent for quantifying labile metal ion levels and dynamics.8 To precisely assess cytosolic labile Zn2+ levels, we used the Zn2+ specific FRET-based biosensor ZapCV2, which consists of a Zn2+-responsive element sandwiched between two fluorescent proteins capable of participating in FRET.9,10 The Zn2+-responsive element in ZapCV2 consists of two zinc fingers from the yeast Zap1 transcription factor. When the Zn2+ concentration increases, the zinc finger domains bind Zn2+ and undergo a conformational change that changes the distance and orientation between the two fluorescent proteins (in this case ECFP and circularly permuted mVenus). In such biosensors, researchers typically measure the FRET channel (excitation of ECFP, emission from cpVenus) and the donor channel (excitation of CFP, emission from CFP) and report the ratio of FRET/CFP. An increase in the FRET ratio (FRET signal / CFP signal) is proportional to an increase in free Zn2+ concentration in the compartment to which the sensor is targeted.

Since sensor expression varies from cell to cell, it is crucial to conduct an in situ calibration of the sensor to accurately quantify labile Zn2+ concentrations in single cells. Calibration of ZapCV2 involves depleting cells of Zn2+ using a metal ion chelator tris(2-pyridylmethyl) amine (TPA) to get the lowest FRET ratio of the sensor in that cell (Rmin), then forcing Zn2+ into the cell by using pyrithione, saponin, and excess Zn2+ to get the highest FRET ratio of the sensor (Rmax). It is critical to optimize in situ calibration conditions for your particular cell type as inaccurate calibration can lead to erroneous estimates of labile Zn2+.11 This protocol will outline the steps for preparing the buffers necessary for a FRET sensor calibration, conducting the calibration in MCF10a cells, and analyzing the data to calculate labile Zn2+ in single cells. While this protocol focuses on MCF10a cells expressing a nuclear excluded version of ZapCV2, the approach should work in any cell type.

Cell culture of MCF10a cells expressing NES-ZapCV2

Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor (3)Timing: 5–7days

  • 1.

    Thaw one vial of MCF10a cells expressing NES-ZapCV2 quickly in a 37°C water bath.

    • a.

      Transfer to a 15mL conical tube containing 5mL of MCF10a media.

    • b.

      Centrifuge at 500×g for 5min.

    • c.

      Aspirate supernatant and resuspend in 10mL of media.

    • d.

      Transfer cell suspension to a 10cm dish.

Note: MCF10a media is composed of 1:1 DMEM/F12 supplement, 5% horse serum, 20ng/mL EGF, 0.5μg/mL hydrocortisone, 100ng/mL cholera toxin, 10μg/mL insulin, and 1% penicillin/streptomycin antibiotics.

  • 2.

    Culture cells in a 10cm dish at 37°C, 5% CO2 and at 90% humidity until 80% confluent.

  • 3.

    Passage cells 1:5 every 2–3days and record passage number.

Alternatives: Researchers may not have access to resources to create stable cell lines. In this case, transient transfection such as lipofectamine or electroporation is a simple way of expressing the NES-ZapCV2 sensor. Please refer to the manufacturer’s protocol for transfection parameters.

Key resources table

Chemicals, peptides, and recombinant proteins
Tris(2-pyridylmethyl) amine 98% (TPA)Sigma-AldrichCat# 723134
Zinc chloride, anhydrous, 99.95% (metals basis)Alfa AesarCat# 87900
Chelex-100, sodium formSigma-AldrichCat# C7901
DMEM/F12, HEPESThermo Fisher ScientificCat# 11330057
Horse serum, New Zealand originThermo ScientificCat# 16050122
HydrocortisoneSigma-AldrichCat# H4001
Gibco EGF recombinant human proteinThermo Fisher ScientificCat# PHG0313
HEPESSigma-AldrichCat# H4034-1KG
Calcium chloride, 99.9%Sigma-AldrichCat# 449709-10G
Potassium chlorideSigma-AldrichCat# P9541-500G
Magnesium chloride hexahydrateSigma-AldrichCat# 63068-250G
Sodium chlorideSigma-AldrichCat# S9625-1KG
Dextrose (D-glucose)Sigma-AldrichCat# D9434-500G
PyrithioneMedChem ExpressCat# HY-B1747
SaponinSigma-AldrichCat# 47036-50G-F
Cholera toxinSigma-AldrichCat# C8052
Potassium hydroxide, ACS reagent >85%Sigma-AldrichCat# 221473-25G
Pen/StrepGibcoCat# 15140–122
0.05% Trypsin-EDTA(1X)GibcoCat# 25300–120
EGTA, molecular biology gradeMilliporeSigmaCat# 324626-25G
Strontium chloride, anhydrous, 99.99+%Sigma-AldrichCat# 439665-25G
Sodium hydroxide pelletsFisher ScientificCat# S318-500
Experimental models: Cell lines
Human: MCF10a+ PB-NES ZapCV2 and PB-H2B- mCherry (stable)Lo etal.5N/A
Software and algorithms
Nikon Elements SoftwareNikon, Inc.N/A
Microsoft ExcelMicrosoft, Inc.N/A
Interactive Shiny AppThis work
Recombinant DNA
pcDNA3.1(+)-NES-ZapCV2 (cpV143) for transient transfectionFiedler etal.9Addgene: #112060
Glass bottom dish 35mm, #1.5 glassibidi81218-200
Nikon Ti-E wide-field microscope (or any wide-field microscope) outfitted with an emission filter wheel capable of detecting FRETNikon, Inc.N/A

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Materials and equipment

HHBSS (PO43- free; pH 7.4)

ComponentFinal concentrationAmount (500mL)
CaCl2 (1 M)1.26mM0.63mL
KCl (1 M)5.4mM2.7mL
MgCl2∗6H2O (1 M)1.1mM0.55mL
NaCl137mM4 g
D-Glucose16.8mM1.5 g
HEPES30mM3.572 g
Chelex-treated waterN/A500mL

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Prepare and adjust pH using NaOH. After sterile filtration, store at 15°C–25°C indefinitely.

HHBSS (PO43-, Ca2+, Mg2+ free; pH 7.4)

ComponentFinal concentrationAmount
KCl (1 M)5.4mM2.7mL
NaCl137mM4 g
D-Glucose16.8mM1.5 g
HEPES30mM3.57 g
Chelex-treated waterN/A500mL

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Prepare and adjust pH using NaOH. After sterile filtration, store at 15°C–25°C indefinitely.

Rmin solution (2X)

ComponentVolumeFinal concentration
HHBSS (PO43- free)9.95mL-
TPA (20mM stock)50μL100μM

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Prepare solution fresh before each calibration.

Rmax solution (2X)

ComponentVolumeFinal concentration
HHBSS (Ca2+ / Mg2+ / PO43- free)9.75mL-
Saponin (0.1 % stock)200μL0.02%
Pyrithione (500μM stock)30μL1.5μM
Solution A100μL-
Solution B100μL-

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Prepare fresh before each calibration.

Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor (4)CRITICAL: To acquire FRET images, the microscope must be able to acquire images for the donor fluorescent protein and the FRET channel nearly simultaneously (a filter wheel will enable images to be collected within ∼ 20ms, moving the dichroic turret will allow images to be collected within ∼ 500ms). The best approach is to use an emission filter wheel to swap between the donor emission wavelength and the acceptor emission wavelength. Researchers should identify whether their microscope setups are able to change filters this quickly.

Note: Many common buffering systems such as phosphate or Tris will interact detrimentally with Zn2+ ions in solution. For example, zinc phosphate is highly insoluble in water, and Tris can act as a chelator of metal ions in solution.12 As such, we use a HEPES-buffered solution, which does not interact with Zn2+ in solution.

Step-by-step method details

Prepare the calibration solutions and buffered Zn2+ solutions

Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor (5)Timing: 4 h

Here, we describe steps for making a buffered Zn2+ solution using the pH jump method,13,14 EGTA as the chelator, and Sr2+ as the counterion. These solutions will be used in the final in situ calibration to deliver controlled levels of labile Zn2+ to the cells during the Rmax phase of the calibration.

  • 1.

    Treat MilliQ water with Chelex-100 to remove any free metal ions in solution.

    • a.

      Add 1 g/L of Chelex-100 to MilliQ water in a plastic container and stir for at least 16 h. Store in plastic bottles indefinitely.

  • 2.

    Soak glassware in metal-free nitric acid for at least 1 h.

    • a.

      Rinse glassware thoroughly with Chelex-treated MilliQ water.

    • b.

      Dry at least 16h on paper towels before use.

Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor (6)CRITICAL: All water in the following steps must be treated with Chelex-100 to remove excess metal ions. Since these solutions are highly precise, small changes in metal ion composition will change the overall free Zn2+ concentration. In addition, all glassware must be washed with nitric acid and rinsed with Chelex-100 treated MilliQ water to remove residual metal ions.

  • 3.

    Make Solution A (EGTA, ZnCl2, and SrCl2 buffered solution).

    • a.

      Weigh out 5mmol of high purity EGTA, 4.5mmol of high purity ZnCl2, and 1.225g KOH pellets.

    • b.

      Combine EGTA, ZnCl2, and KOH in a clean metal-free beaker and add 15mL Chelex-treated MilliQ water.

    • c.

      Heat slightly while stirring. Add 1.0M KOH in Chelex-treated water incrementally until pH is between 7 and 8.5.

    • d.

      Prepare a 50mL 1.0M ZnCl2 solution in Chelex-treated water in a volumetric flask.

    • e.

      Prepare 50mL of a 1.0M SrCl2 solution in Chelex-treated water using a volumetric flask.

    • f.

      Note the pH of the Zn-EGTA solution to the thousandths place.

    • g.

      Add a known number of mmol of ZnCl2 solution by pipetting between 20‒40μL of the ZnCl2 solution into the Zn-EGTA solution.

      Note: While adding ZnCl2 solution, make sure to dip the pipette tip into the solution and mix it properly. Always use a new pipette tip for mixing.

    • h.

      Calculate the ΔpH/ΔZn. This is the starting value.

    • i.

      Add the ZnCl2 solution, record the pH and the ΔpH/ΔZn with each addition of ZnCl2.

    • j.

      Add aliquots of the KOH solution as necessary to keep the pH within the 7–8.5 regime. An example titration is shown in Table 1.


      Example buffered Zn2+ titration

      Added KOH/ZnCl2 (mmol)pHΔpHΔpH/ΔZn
      + 0 ZnCl27.47
      + 20 ZnCl27.270.210
      + 40 KOH7.46
      + 20 ZnCl27.20.210
      + 40 KOH7.45
      + 20 ZnCl27.270.189
      + 40 KOH7.45
      + 20 ZnCl27.260.199.5
      + 40 KOH7.43
      + 20 ZnCl27.250.189
      + 40 KOH7.42
      + 40 ZnCl27.100.328
      + 100 KOH7.51
      + 40 ZnCl27.180.338.25
      + 100 KOH7.61
      + 40 ZnCl27.260.358.75
      + 100 KOH7.72
      + 40 ZnCl27.330.399.75
      + 100 KOH7.80
      + 40 ZnCl27.430.379.25
      + 0 KOH7.43
      + 40 ZnCl27.150.287
      + 80 KOH7.42
      + 40 ZnCl27.170.256.25
      + 100 KOH7.53
      + 40 ZnCl27.260.276.75
      + 100 KOH7.64
      + 40 ZnCl27.360.287
      + 80 KOH7.66
      + 40 ZnCl27.420.266.5
      + 80 KOH7.71
      + 40 ZnCl27.470.246
      + 80 KOH7.77
      + 20 ZnCl27.550.225.5

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    • k.

      When the ΔpH/ΔZn is less than ½ the original value, the titration is complete.

    • l.

      Add aliquots of the KOH solution until the pH of solution A is 7.0, then transfer the titrated solution to a 50mL volumetric flask.

    • m.

      Add 10.0mL of the SrCl2 solution and bring the volume of the solution to the 50mL mark with Chelex-treated water.

    • n.

      Transfer the solution to a clean plastic container. This solution can be stored at room temperature (15–25°C) indefinitely.

      Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor (7)CRITICAL: All chemicals contain impurities. Due to the precise nature of this titration, researchers must consider impurity when measuring chemical masses. For example, if the EGTA used is 95% purity, the mass used will be 1.05 times the “ideal” mass of 5mmol 100% purity EGTA.

      Note: The pH must be in the neutral range for EGTA to dissolve completely. This will require heating and stirring to dissolve completely. A slightly acidic pH may be required for dissolving ZnCl2.

  • 4.

    Make Solution B (EGTA and SrCl2 buffered solution).

    • a.

      Weigh out 5mmol of high purity EGTA.

    • b.

      Weigh out 1.2g of KOH pellets.

    • c.

      Place in a clean, dry flask and add 10.0mL of 1.00M SrCl2 solution. Note the pH.

    • d.

      Add aliquots of 1.0M KOH until the pH is 7.0.

    • e.

      Transfer solution to a clean, dry volumetric flask and bring volume to 50mL with Chelex-treated MilliQ water.

    • f.

      Transfer to a clean dry plastic container. This solution can be stored at room temperature (15–25°C) indefinitely.

Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor (8)Pause point: Solutions A and B may be prepared well in advance and can be stored at 15°C–25°C indefinitely.

Note: The ratio of solution A and solution B in Rmax may need to be optimized for your cell line, but a good starting place is a 5:5 ratio of A:B (Figure1C).

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Buffered Zn2+ system

(A) Example of a two-ion buffering system with EGTA as the chelating agent, and Sr2+ as the counterion. Varying Sr2+ concentration will act to sequester EGTA to tune free Zn2+ in the buffer.

(B) Components of solutions A and B (top) and the range of free Zn2+ that can be achieved using this buffering system (C) A chart illustrating varying Solution A:Solution B ratios and their corresponding free Zn2+ concentrations at pH 7.0 in the final Rmax buffer. Figurecreated with BioRender.

Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor (10)CRITICAL: Simply weighing out the masses of each chemical and salt will result in a large error, and therefore an imprecise free Zn2+ concentration in the final buffer. In order to make these solutions, we use a modified version of the pH jump method.13,14 When Zn2+ binds to EGTA at neutral pH, it displaces two H+ ions, which lowers the pH. Since H+ concentration can be very precisely calculated using a pH meter, we use pH as a readout for the titration. We then titrate in Zn2+ and record the pH to monitor the titration progression. We maintain the pH of the solution between 7‒8 using potassium hydroxide as needed. As the Zn2+ concentration approaches the EGTA concentration, the number of H+ ions it displaces will decrease, indicating that the Zn2+ concentration is within the buffering regime of EGTA (Table1). The addition of Sr2+ acts as a counterion to sequester EGTA to tune the free Zn2+ concentration to the desired value. It is important to use plastic containers to store components, since glass can leach and retain metal ions. In addition, chemical weights must be corrected for the purity of the chemical to ensure precise molarity.

Note: It is necessary to ensure a stable concentration of labile Zn2+ throughout the Rmax phase of the calibration. To do this, we use a buffer system that has a precise Zn2+ concentration, which has been shown to yield more accurate Rmax values during the final stage of the calibration than high concentrations of unbuffered zinc.11 We combine a solution of ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) Zn2+ and Sr2+ (Solution A) with a solution of Sr2+ and EGTA (Solution B) (Figures1A and 1B). The ratio of these two solutions will determine the concentration of free Zn2+ in the Rmax buffer (Figure1C). It is important to note that any chelator-counterion pair can be used in this system to accurately tune the free Zn2+ as long as the affinity of the counterion to the chelator is less than that of Zn2+. It is also crucial that solutions A and B are at the exact same pH, since free Zn2+ in these solutions is pH dependent. For most cell lines, we use Sr2+ and EGTA as the chelator-counterion pair since this pair can buffer Zn2+ between 8nM and 1.1μM at pH 7.0. Another option for an ion-counterion-chelator system is Zn2+/Ca2+/EGTA.

Note: To precisely calculate the free metal concentration of this two-ion buffer system, we use a set of equations, established apparent dissociation constants for each metal/chelator pair (KDMLapp), ionic strengths, and pKa values for the chelators at a constant temperature and pH:











where M2 is the metal whose free concentration we are varying (in this case Zn2+), M1 is the competing metal with lower affinity for the chelator (in this case Sr2+), and L is the chelator (in this case EGTA). Additionally, any variable with a subscript “T” refers to the total concentration of that species in solution. These equations and sample calculations are presented in TableS1. For our purposes, we will be using the affinity constants of EGTA for Zn2+ (12.6) and Sr2+ (8.43) at 25°C, as well as the two relevant pKa values for EGTA (pKa1= 9.40, pKa2= 8.78) at 25°C. TableS2 presents the Kd and pKa values for a series of metal chelators. Since all KDMLapp values were determined empirically at ionic strength 0.1 M, we will correct the KDMLapp to reflect this different ionic strength and solution pH in Equation4. For this protocol, all of the calculations have been done assuming free Zn2+ as M2 and Sr2+ as M1, and free Zn2+ values are available in Figure1C. It is worth noting that the KDMLapp calculation assumes two relevant pKa values. If your chelator has only one, refactor Equation4 to reflect this. Note that Equation5 contains [M2], which is the unknown value for which we are solving. To resolve this, we include an initial “guess” of the free [M2], then iteratively calculate [M2] based on the initial guess until the value converges on the true [M2] value. We have attached an interactive spreadsheet in supporting information that contains logK and pKa values of commonly used chelators and counterions as well as example calculations. In addition, we have deployed an interactive Shiny App to easily calculate the range of free metals values for several chelator/ion/counterion systems (

Live cell in situ calibration of the cytosol-targeted ZapCV2 sensor

Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor (11)Timing: 2h (plus cell plating and recovery)

Here, we outline steps to conduct an in situ calibration of the NES-ZapCV2 sensor in order to calculate the concentration of resting labile cytosolic Zn2+. This is done by first imaging the cells in their native resting state, then depleting labile Zn2+ from the cells using a membrane permeable chelator TPA. The media is changed, and cells are treated with buffered Zn2+, pyrithione, and saponin, which floods the cells with excess Zn2+ and saturates the sensor (Table2, Methods video S1).

  • 5.

    Trypsinize MCF10a cells expressing the NES-ZapCV2 sensor.

    • a.

      Remove the 10cm maintenance dish from the incubator, aspirate off the media and wash the cells two times with 7–10mL of 1X PBS. Cells should be 80% confluent and will yield approximately 5 million cells.

    • b.

      Add 1mL of 0.05% trypsin-EDTA to the dish and allow to incubate for 15min at 37°C in the tissue culture incubator.

    • c.

      Quench the trypsin with 8mL of growth media and wash the plate with the cell suspension to remove remaining attached cells.

    • d.

      Centrifuge cell suspension at 500×g for 5min to pellet.

    • e.

      Resuspend cells in 1mL growth media and count using a hemocytometer or an automated cell counter.

  • 6.

    Plate 300,000 cells per 35mm dish in 1mL treatment media.

  • 7.

    Allow to adhere to the dish at least 16h in the tissue culture incubator.

  • 8.

    Thirty minutes before the imaging experiment, remove one dish from the tissue culture incubator.

  • 9.

    Wash the imaging dish three times with 2mL of imaging media (HHBSS, PO43- free pH 7.4).

  • 10.

    Aspirate media and replace with 1mL of fresh imaging media.

  • 11.

    Allow the dish to sit at room temperature (20–25°C) for 30min.

Note: Do not place the dishes back in the tissue culture incubator. HEPES buffer cannot buffer CO2 and will quickly acidify and kill the cells.

Note: The imaging media must be equilibrated to room temperature when washes and imaging take place. Perform this imaging experiment at room temperature. Fluctuations in temperature can cause changes in the FRET response.

  • 12.

    Make HHBSS (both PO43-, Ca2+, Mg2+ free, and PO43- free), 2X Rmin solution, and 2X Rmax solution as defined in the materials and equipment section.

  • 13.

    Set up acquisition parameters while the imaging dish is equilibrating.

Note: Depending on the acquisition software available, researchers may need to manually acquire images at these time points or set up an automatic acquisition. There are three phases to the calibration Rrest (resting state of ZapCV2), Rmin (unbound state of ZapCV2) and Rmax (bound state of ZapCV2) as described in Methods video S1.

  • 14.

    Place cells on the microscope using a 35mm dish holder, ensuring the metal arms hold the dish firmly in place.

Note: During the calibration, there is a high chance of moving the dish during buffer addition and washing which can result in the loss of the field of view and therefore the calibration of that dish. To minimize the odds of bumping the dish, use a 35mm dish holder with metal arms to keep the dish firmly in place. While removing solution from the imaging dish try to stabilize the dominant hand with other hand and try to avoid touching the base of dish with the pipette tip.

  • 15.

    Switch to a 20X air objective and focus the cells using the donor channel (in this case, CFPex, CFPem), ensuring that the field of view contains multiple cells (problem 1).

  • 16.

    Draw regions of interest (ROI) in the cytosol of each cell, in addition to at least three regions of interest in the empty space between cells, which will be recorded as background (Methods video S1).

Optional: You may acquire the calibration before drawing the ROIs. It is suggested that you draw ROIs to monitor sensor response throughout the calibration. Adding an ROI and tracking the signals across time will be different in each acquisition software. Consult the user’s manual for more information.

  • 17.

    Monitor the donor channel (CFPex, CFPem) and the FRET channel (CFPex, YFPem) and ensure the exposure times are the same for both channels.

  • 18.

    Ensure the optical configurations are set as follows:

    • a.

      CFP Ex: 440/20, 455 dichroic, Em: 480/20.

    • b.

      CFP/YFP FRET Ex: 440/20, 455 dichroic, Em: 540/21.

Optional: YFP Ex: 510/25, 518 dichroic, Em: 540/21.

  • 19.

    Begin the first phase Rrest and monitor for at least 5min and ensure that the FRET ratio does not change appreciably before beginning the next phase of the calibration.

    • a.

      If the resting FRET ratio is unstable for 5min, the Rrest phase can be extended until the FRET ratio is stable (Methods video S1).

  • 20.

    Pause the acquisition and add 1mL of the 2X Rmin solution (final concentration of 50μM TPA).

  • 21.

    Resume the acquisition.

  • 22.

    Monitor the Rmin for at least 3min and ensure that the Rmin value is not changing before beginning the next phase of the calibration (problem 2).

Note: Upon addition of Rmin solution, there will be a sudden drop in FRET ratio and then Rmin value will remain unchanged. The donor channel (YFP) will not change appreciably upon addition of the Rmin solution (Figures2B and 2C, Methods video S1).

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In situ calibration of NES-ZapCV2 in HeLa and MCF10a cells

(A) Sample FRET ratio image of cells in Rrest buffer, after exposure to Rmin buffer, and after exposure to Rmax buffer in both HeLa and MCF10a cells. Scale bar= 60μm.

(B) A plot of the average FRET ratio of cells after each perturbation in both MCF10a and HeLa cells. Phases of the calibration are labeled above the curves. Error bars represent 95% confidence intervals. n= 7 cells.

(C) The corresponding CFP, YFP, and FRET intensities during each phase of the calibration. Phases of the calibration are labeled above the data points. Figurecreated with BioRender.

  • 23.

    Pause the acquisition.

  • 24.

    Remove as much of the calibration solution as possible making sure not to touch the dish with the pipette tip.

  • 25.

    Quickly wash twice with 1mL of imaging buffer (HHBSS, PO43-, Ca2+, Mg2+ free; pH= 7.4).

  • 26.

    Remove the buffer each time and resupply with 1mL of imaging buffer to the dish.

  • 27.

    Start acquiring the Rmax phase of the calibration and add 1mL of Rmax buffer.

  • 28.

    Collect one frame every 10s until the signal peaks and begins to decline (Figures1B and 1C) (problem 2, problem 3).

Note: The donor channel (YFP) will not change appreciably upon addition of the Rmax solution but may decrease slightly due to photobleaching from a higher acquisition rate (Figures2B and 2C, Methods video S1).

Note: The Rmax buffer will require optimization in new cell lines. If the FRET ratio peaks veryquickly and immediately drops off after adding the Rmax it is likely that the true Rmax has not been achieved. Therefore, researchers will need to change concentrations of pyrithione, saponin, or the ratio between solutions A and B to yield a steady rise and constant plateau in FRET signal after Rmax addition (Figure2B). Using high concentrations of unbuffered Zn2+ is not encouraged, since this can lead to a quick plateau and decay. For more information and examples of reagent optimization, refer to Carter etal., 2017, Analytical Chemistry.11

Note: During Rmax, cells will begin to change shape and peel off the dish and become apoptotic due to the pyrithione, Zn2+ and Sr2+ in the Rmax solution. This is normal. Once cell death becomes apparent, end the acquisition. The cells cannot be calibrated again.

  • 29.

    Save each time-lapse image in either the native format (.nd2, .rwl, .orf) or as a TIFF stack (.tif) for downstream analysis.


Phases and imaging frequencies for the in situ calibration

PhaseTreatmentCapture frequencyImaging duration
RrestUnperturbed cells in a resting state1 frame every 30 s5–10min
RminTreatment of cells with a Zn2+ chelator1 frame every 30 s5min
RmaxTreatment of cells with buffered Zn2+, an ionophore, and a detergent1 frame every 10 s5–20min

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Methods video S1. Example calibration and analysis parameters, related to “Live cell insitu calibration of the cytosol-targeted ZapCV2 sensor,” “Image processing and analysis,” and “Limitations” steps of the protocol:


Click here to view.(87M, mp4)

Image processing and analysis

Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor (13)Timing: 30min

This step enables researchers to extract FRET ratios of each cell, background correct the FRET signal, and calculate the labile Zn2+ ion concentration in cells.

  • 30.

    Open one time-lapse image in ImageJ/Fiji.

  • 31.

    Draw ROIs by selecting the “Oval ROI” tool.

    • a.

      Draw several ROIs in different cells, as well as at least one ROIs in an area of the image devoid of cells (this will serve as a background ROI).

    • b.

      After drawing each ROI, right click and select “Add to ROI Manager.” A new window will pop up and list your ROIs.

    • c.

      Alternatively, select Analyze>Tools>ROI Manager… to open the ROI manager and manually add each ROI by pressing the hotkey “t”.

Note: Since cells may move or change shape over time, ensure that the region covers the cytosol of one cell throughout the time lapse image.

  • 32.

    Select Analyze>Set Measurements… and ensure that only “mean gray value” is selected.

  • 33.

    Deselect all other measurements.

  • 34.

    In the ROI manager, select More>Multi Measure.

  • 35.

    Check “Measure all X slices” and “One row per slice” then click OK.

  • 36.

    Copy data to an Excel spreadsheet.

    • a.

      Measurements from each ROI at one frame are listed on the rows of the table, and each ROI is listed in each column.

    • b.

      The measurements of one channel are in rows 1, 3, 5, etc. and the other are in 2, 4, 6, etc.

  • 37.

    Subtract the background intensities at each timepoint from the intensities of the test ROIs.

Note: The background intensities will change over the time lapse, so ensure the background is subtracted with its time-paired ROI.

  • 38.

    Calculate the FRET ratio by dividing the background-subtracted FRET channel by the background-subtracted donor channel.

  • 39.

    Find the mean Rrest ratio of each ROI by averaging the FRET ratios during the Rrest phase of the calibration.

Note: If the Rrest was unstable (increasing or decreasing), only average the FRET ratios for the region of the curve that was stable, typically right before changing to the Rmin solution.

  • 40.

    Find the mean Rmin ratio of each ROI by averaging the FRET ratios for the last few data points of the Rmin phase of the calibration.

  • 41.

    Find the Rmax by finding the maximum FRET ratio during the Rmax phase of the calibration.

  • 42.

    Use the following equations to calculate the [Zn2+]cytosol for each ROI, where Kd= 5.3nM, and n= 0.2910, which were determined empirically for this specific sensor, ZapCV2, for Zn2+.



Expected outcomes

The labile Zn2+ pool is highly dynamic in cells and can be measured using a FRET-based fluorescent biosensor. In this protocol, we describe steps to perform an in situ calibration of this biosensor using a low and high Zn2+ solution to find the dynamic range of the sensor. Depending on the available Zn2+ in the growth media, MCF10a cells can have a labile Zn2+ concentration of between 1 pM and 1nM, with an average of 80 pM in Zn2+ adequate media (Figure3C). This protocol can be used to determine the resting labile Zn2+ concentration in treated or untreated cells, or in a variety of cell types.

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Image processing and analysis examples

(A) Average traces of FRET ratio throughout an insitu calibration for both MCF10a and HeLa cells. Rrest, Rmin, and Rmax are labeled on the graph.

(B) The equation for calculating labile Zn2+ using the Rrest, Rmin and Rmax parameters, the Hill coefficient (n)and the sensor’s apparent Kd.

(C) Quantification of resting cytosolic Zn2+ concentration in MCF10a and HeLa cells using the equation in (B) Error bars represent the 95% confidence interval. Figurecreated with BioRender.


Sensor limitations

The ZapCV2 sensor was optimized in HeLa cells and has been used in several cell types such as MCF10a cells, MDA-MB-231 cells, primary hippocampal neurons, and primary macrophages.1,15,16 We have found that the calibration solutions outlined in this proposal work for most cell types. That said, researchers may need to optimize the calibration conditions (see problem 2 and problem 3 below). In HeLa cells, the sensor dynamic range is 1.7–2.510, in MCF10a cells the dynamic range is typically 1.4–1.71. If the dynamic range is less than 1.35, the Zn2+ concentration is often overestimated.17 We recommend limiting analysis to cells in which the dynamic range is over 1.4 (Methods video S1). The sensor expression can vary from cell to cell and the expression level can impact the dynamic range and therefore the signal to noise ratio of the sensor. We recommend avoiding very bright and very dim cells in the analysis.

Microscope limitations

Imaging FRET sensors comes with its own challenges. First, the most important part of the microscope setup is the ability to acquire the donor channel and the FRET channel almost simultaneously. The optimal system involves the use of an emission filter wheel, which is not as common as filter turrets that hold filter cubes. Ensuring the capability of your microscope to capture FRET is critical for the success of this protocol. Any perturbation in temperature can influence FRET efficiency, so it is important that cells are always kept at room temperature (20–25°C) and are allowed to equilibrate to atmospheric conditions before the calibration.


Problem 1

Not enough cells in the imaging dish for calibration experiment.

Potential solution

Insufficient adhesion of cells to the imaging dish may result in their detachment during multiple washing steps. It would be advisable to allow the cells to adhere for an additional day after plating. If the cell type being studies is only weakly adherent, coating the imaging dish with poly-lysine, collagen or gelatin can increase adhesion.

Problem 2

Poor dynamic range of the sensor during Rmin and Rmax phases of the calibration.

Potential solution

Optimize the chemical components in the Rmax solution. Pyrithione is an ionophore that enables Zn2+ to pass through the plasma membrane and intracellular membranes. But pyrithione can be quite toxic to cells and high concentrations tend to give poor in situ calibrations.11 In addition, we find that the concentration of Zn2+ may need to be optimized. This protocol outlines the use of a solution in which Zn2+ is buffered at 101.4nM. In general, we have found that buffered Zn2+ solutions in the nM range give better calibrations than high concentrations of unbuffered Zn2+.11 While not strictly necessary for Zn2+ to enter a cell, we have found that low concentrations of saponin, a membrane permeabilizing detergent, often yield a higher dynamic range. The concentration of saponin may need to be adjusted for different cell types.

Problem 3

The Rmax step peaks very quickly and decays without a plateau.

Potential solution

When the Rmax peaks and rapidly decays, it is likely the true Rmax of the sensor has not been achieved. This requires optimizing pyrithione, saponin, and the buffered zinc solution. We often find that the rapid peak and decay is a sign of the cells dying very rapidly in the Rmax solution conditions. Therefore, we advise lowering the pyrithione concentration and lowering the concentration of buffered Zn2+ to try to achieve a slower rise and plateau. If it is not possible to achieve a full calibration (Rmin followed by Rmax), you can conduct two “half calibrations” in two different dishes. This involves using one dish to get the Rmin value, by first measuring Rrest, followed by Rmin. In the other dish, measure the Rrest, then add the Rmax solution to obtain the Rmax value.

Resource availability

Lead contact

Further information and requests for resources should be directed to the lead contact, Amy Palmer (ude.odaroloc@remlap.yma).

Technical contact

Requests for further technical information should be directed to the technical contact, Amy Palmer (ude.odaroloc@remlap.yma).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate or analyze any new datasets or code.


We thank the University of Colorado Biochemistry Cell Culture Core (specifically Theresa Nahreini) for assistance with cell culture. We would like to acknowledge NIGMS MIRA R35 GM139644 (A.E.P.) for generous financial support.

Author contributions

Conceptualization and methodology, A.E.P., A.R., and S.E.H.; investigation, A.R. and S.E.H.; writing, S.E.H. and A.E.P.; funding acquisition, supervision, and project administration, A.E.P.

Declaration of interests

The authors declare no competing interests.


Supplemental information can be found online at

Supplemental information

TableS1. Free zinc calculator, related to the step of “Prepare the calibration solutions and buffered Zn2+ solutions”:

Click here to view.(120K, xlsx)

TableS2. Kd and pKa values of metal chelators, related to the step of “Prepare the calibration solutions and buffered Zn2+ solutions”:

Click here to view.(12K, xlsx)


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Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor (2024)
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