使用基因编码荧光传感器 HyPer7 测量完整人体细胞中的双氧水水平

Lianne J. H. C. Jacobs; Michaela N. Hoehne; Jan  Riemer

doi:10.21769/BioProtoc.4538

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Peer-reviewed

Measuring Intracellular H₂O₂ in Intact Human Cells Using the Genetically Encoded Fluorescent Sensor HyPer7

使用基因编码荧光传感器 HyPer7 测量完整人体细胞中的双氧水水平

LJ Lianne J. H. C. Jacobs

MH Michaela N. Hoehne

JR Jan Riemer email

发布: 2022年10月20日第12卷第20期 DOI: 10.21769/BioProtoc.4538 浏览次数: 2358

评审: David PaulVsevolod BelousovAnonymous reviewer(s)

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Abstract

Depending on its local concentration, hydrogen peroxide (H₂O₂) can serve as a cellular signaling molecule but can also cause damage to biomolecules. The levels of H₂O₂ are influenced by the activity of its generator sites, local antioxidative systems, and the metabolic state of the cell. To study and understand the role of H₂O₂ in cellular signaling, it is crucial to assess its dynamics with high spatiotemporal resolution. Measuring these subcellular H₂O₂ dynamics has been challenging. However, with the introduction of the super sensitive pH-independent genetically encoded fluorescent H₂O₂ sensor HyPer7, many limitations of previous measurement approaches could be overcome. Here, we describe a method to measure local H₂O₂ dynamics in intact human cells, utilizing the HyPer7 sensor in combination with a microscopic multi-mode microplate reader.

Graphical abstract:

Overview of HyPer7 sensor function and measurement results.

Keywords: H₂O₂measurement (H₂O₂测量)

HyPer7 (HyPer7)

Real-time imaging (实时成像)

Subcellular resolution (亚细胞分辨率)

pH-independent (非pH依赖)

Background

Aerobic life comes at a price due to the formation of reactive oxygen species (ROS) as an unavoidable by-product of essential metabolic processes. ROS is a collective term for reactive chemical species containing oxygen, of which hydrogen peroxide (H₂O₂) is the most relevant member due to its long half-life and comparably high cellular concentration. H₂O₂ has both beneficial and deleterious effects on cells depending on its levels, flux, and the functional state of the cell (Sies and Jones, 2020). Consequently, cellular H₂O₂ levels are tightly controlled.

The subcellular dynamics of H₂O₂ are poorly understood in intact mammalian cells. To explore these dynamics, biosensors that are targetable, sensitive, specific, and reversible are required. Research previously relied on small chemical probes (mostly irreversible and therefore not measurements of dynamic fluxes of H₂O₂) or comparably insensitive genetically encoded sensors (reversible but insensitive) (Roma et al., 2018; Kalinovic et al., 2019; Liao et al., 2020; Plecita-Hlavata et al., 2020).

In recent years, more sensitive genetically encoded fluorescent H₂O₂ sensors were developed, including HyPer7 (Pak et al., 2020). HyPer7 consists of a circular permutated yellow fluorescent protein (cpYFP) that is integrated into the H₂O₂-sensing regulatory domain of Neisseria meningitidis OxyR (Oxy-RD). OxyR is a natural H₂O₂ sensor and transcription factor. OxyR from different organisms have different sensitivities; the specific Oxy-RD domain from N. meningitidis renders HyPer7 particularly sensitive and allows measurement of close to baseline H₂O₂ levels in otherwise unperturbed mammalian tissue cell lines.

Oxy-RD contains two cysteine residues that react in a sensitive and highly specific manner with H₂O₂ and consequently form a disulfide bond. This disulfide bond in Oxy-RD puts a strain on the fused cpYFP, thus changing the cpYFP excitation spectrum. cpYFP has two excitation maxima at ca 400 nm and 499 nm, respectively, and emits at 516 nm. Changes in the sensor oxidation state result in ratiometric changes with decreases in the excitation maximum at 400 nm and increases at 499 nm upon Oxy-RD oxidation (Pak et al., 2020). Notably, targeted mutations in the cpYFP backbone result in pH insensitivity of HyPer7. This is a clear advantage compared with previous HyPer sensor variants (e.g., HyPer3) that were all pH sensitive in the physiological pH range and required laborious pH control experiments.

Sensors for different biomolecules can be combined with different treatments of cells, with different genetic backgrounds, or with genetic engineering tools. One such genetically encoded tool is D-amino acid oxidase (DAO), an enzyme that converts D-amino acids to the corresponding α-keto acid and generates as a by-product H₂O₂ (Matlashov et al., 2014). Since DAO can be targeted to different regions within a cell, it can be employed for the localized generation of H₂O₂.

Here, we describe a protocol to measure local H₂O₂ dynamics in cytosol and mitochondria. Combining this method with genetically engineered tools, such as the DAO system or CRISPR-cas9–mediated gene editing, to specifically remove parts of the antioxidative system allows detailed investigations of subcellular H₂O₂dynamics.

Materials and Reagents

Cultivation of HEK293 cells
1. 1.5 mL Eppendorf tube (Diagonal, catalog number: 02-023-0100)
2. Sterile filter pipette tips:
  10 µL (Greiner Bio-One, Sapphire, catalog number: 772353)
  100 µL (Greiner Bio-One, Sapphire, catalog number: 774353)
  1,250 µL (Greiner Bio-One, Sapphire, catalog number: 778353)
3. 96-well plate (µClear, Greiner Bio-One, catalog number: 655090)
4. 15 mL Falcon tube (VWR, catalog number: 734-0451)
5. LUNA reusable cell counting slide (Logos Biosystems, catalog number: L12011)
6. Autoclaved disposable glass Pasteur pipettes without cotton pad (VWR, catalog number: HECH40567001)
7. Sterile, individually packed serological pipettes:
  5 mL pipettes (Sarstedt, catalog number: 86.1253.001)
  10 mL pipettes (Sarstedt, catalog number: 86.1254.001)
8. 10% Fetal calf serum (FCS, Sigma-Aldrich, catalog number: F0804)
9. 1% Penicillin/streptomycin (P/S, Sigma-Aldrich, catalog number: P0781-100ML)
10. Dulbecco's Modified Eagle Medium high glucose (DMEM, Thermo Fisher, Gibco, catalog number: 41965062)
11. Reagent reservoir (VWR, catalog number: 613-1175)
12. Flp-In T-Rex HEK293 cells (Human embryonic kidney 293, Invitrogen, catalog number: R78007)
13. Poly-L-lysine (Sigma-Aldrich, catalog number: P4832-50mL)
14. Dulbecco's Phosphate Buffered Saline powder (DPBS, Sigma-Aldrich, catalog number: D5652)
15. 10× Trypsin-EDTA solution (Sigma-Aldrich, catalog number: T4174)
16. Trypan Blue (Logos Biosystems, catalog number: T13001)
17. DMEM medium-complete (Gibco, catalog number: 41965039) (see Recipes)
18. DPBS (see Recipes)
19. Trypsin-EDTA solution (see Recipes)
Transfection of HEK293 cells
1. Plasmids:
  pCS2+HyPer7-NES [addgene: plasmid #136467 (Pak et al., 2020)]
  pCS2+MLS-HyPer7 [addgene: plasmid #136470 (Pak et al., 2020)]
2. Polyethylenimine (PEI) (Polysciences, catalog number: 23966-1)
3. Polyethylenimine (PEI) 1 mg/mL (see Recipes)
4. Dulbecco’s Modified Eagle Medium medium-pure (DMEM, Thermo Fisher, Gibco, catalog number: 41965062) (see Recipes)
Induced expression of DAO in HEK293 cells
1. Doxycycline (DOX) (AppliChem, catalog number: A2951,0005)
H₂O₂ measurement
1. D-Alanine (Sigma-Aldrich, catalog number: 338-69-2)
2. L-Alanin BioChemica (Applichem, catalog number: A3690,0100)
3. H₂O₂ (Sigma-Aldrich, catalog number: 216763-100ML)
4. Minimal media (see Recipes):
  NaCl (Roth, catalog number: 7647-14-5)
  KCl (Roth, catalog number: 7447-40-7)
  MgCl₂ (Roth, catalog number: 2189.2)
  CaCl₂ (Merck, catalog number: 23.891.000)
  HEPES (VWR, catalog number: 7365-45-9)
  Glucose (CIL, catalog number: 110187-42-3)
  10% Fetal calf serum (FCS, Sigma-Aldrich, catalog number: F0804)

Equipment

Cultivation of HEK293 cells
1. Laminar flow hood class II (ENVAIR eco)
2. CO₂ incubator (New Brunswick)
3. Vacuum pump (laboport)
4. Microscope (motic AE2000)
5. Multichannel pipette (VWR)
6. Cell counter – LUNA-II (Logos Biosystems, catalog number: L40002)
Equipped cell culture laboratory containing, e.g., a laminar flow hood class II (ENVAIR eco), a CO₂incubator for cultivation of cells (with cultivation set at 37 °C and 5% CO₂), a vacuum pump for removal of medium using sterile glass Pasteur pipettes, a microscope, a cell counter, and a cooling centrifuge.
Transfection of HEK293 cells
In addition to A.
1. Vortex shaker
H₂O₂ measurement
1. Cytation 3 (Agilent, BioTek)
  CO₂ control
  Injection system (optional)
  390LED Rev H (Agilent, BioTek, catalog number: 1225009)
  roGFPsmall filterblock ex.390 em. 525 Rev D (Agilent, BioTek, catalog number: 1225108)
  465LED Rev I (Agilent, BioTek, catalog number: 1225001)
  GFP filterblock ex. 469 em. 525 Rev I (Agilent, BioTek, catalog number: 1225101)
2. Minimal media [HEPES buffer (HBSS) solution from (Poburko et al., 2011)] (see Recipes)
3. Complete minimal media [HEPES buffer (HBSS) solution from (Poburko et al., 2011)] (see Recipes)

Software

Redox Ratio Analysis (RRA) [Dr M.D. Fricker, https://markfricker.org/77-2/software/redox-ratio-analysis/ (Fricker, 2016)]
R (https://www.r-project.org/)
Rstudio (https://www.rstudio.com/)

Procedure

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Jacobs, L. J. H. C., Hoehne, M. N. and Riemer, J. (2022). Measuring Intracellular H₂O₂ in Intact Human Cells Using the Genetically Encoded Fluorescent Sensor HyPer7. Bio-protocol 12(20): e4538. DOI: 10.21769/BioProtoc.4538.