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. Author manuscript; available in PMC: 2024 Nov 1.
Published in final edited form as: Exp Eye Res. 2023 Oct 2;236:109653. doi: 10.1016/j.exer.2023.109653

A simplified protocol to induce hypoxia in a standard incubator: a focus on retinal cells

Bhavneet Kaur a, Bruna Miglioranza Scavuzzi a, Steven F Abcouwer a, David N Zacks a
PMCID: PMC10732591  NIHMSID: NIHMS1943682  PMID: 37793495

Abstract

Hypoxia chambers have traditionally been used to induce hypoxia in cell cultures. Cellular responses to hypoxia can also be mimicked with the use of chemicals such as cobalt chloride (CoCl2), which stabilizes hypoxia-inducible factor alpha-subunit proteins. In studies of ocular cells using primary cells and cell lines, such as Müller glial cell (MGC) lines, photoreceptor cell lines, retinal pigment epithelial (RPE) cell lines and retinoblastoma cell lines oxygen levels employed in hypoxia chambers range typically between 0.2% and 5% oxygen. For chemical induction of hypoxic response in these cells, the CoCl2 concentrations used typically range from 100 to 600 μM. Here, we describe simplified protocols for stabilizing cellular hypoxia-inducible factor-1α (HIF-1α) in cell culture using either a hypoxia chamber or CoCl2. In addition, we also provide a detailed methodology to confirm hypoxia induction by the assessment of protein levels of HIF-1α, which accumulates in response to hypoxic conditions. Furthermore, we provide a summary of conditions applied in previous studies of ocular cells.

Keywords: Hypoxia chamber, cobalt chloride, hypoxia-inducible factor, retina

1. Introduction

Normal oxygen levels in different body tissues vary significantly (McKeown, 2014). The term “normoxia” describes the typical oxygen partial pressure of O2 in media surrounding cell cultures under atmospheric oxygen conditions, which is 20.9% oxygen in dry air (Wenger et al., 2015) and depends on factors such as altitude and humidity (Carreau et al., 2011; McKeown, 2014; Wenger et al., 2015). “Hypoxia” describes decreased or insufficient oxygen supply to cells, tissues, or organs, compared to physiological conditions (Carreau et al., 2011; McKeown, 2014; Wenger et al., 2015). The retina is one of the most metabolically active tissues in the body and requires regular and constant oxygen supply for the maintenance of its function (Wangsa-Wirawan and Linsenmeier, 2003).

Conditions such as retinal detachment, diabetes, occlusion of the central retinal artery, or thrombosis of the central retinal vein can compromise oxygenation, leading to the progression of retinal diseases and cell death (Alder et al., 1997; Curtis et al., 2009; Grimm and Willmann, 2012; Kaur et al., 2008; Ross et al., 2022). Retinal detachment (RD) is a serious ocular pathology that occurs when the neurosensory retina becomes separated from the underlying retinal pigmented epithelium (RPE) and the choroid, the vascular layer that provides blood supply and oxygen to the retina. When RD occurs, proper oxygen supply to retinal cells is impaired, leading to hypoxia in the affected area (Piccolino et al., 2005). Hypoxia, in turn, initiates a sequence of pathological events within the retina, encompassing the activation of hypoxia-inducible factor 1-alpha (HIF-1α) and the subsequent upregulation of various stress-related genes, promoting inflammation, augmenting reactive oxygen species (ROS) production, and leading to photoreceptor degeneration (Campochiaro, 2015; Shinojima et al., 2021). In diabetes, for example, chronic hyperglycemia has been shown to damage blood vessels in the retina, leading to vascular constriction and reduced oxygen delivery, even in very early stages of the disease (Alder et al., 1997). This, in turn, prompts the release of vascular endothelial growth factor (VEGF), promoting abnormal blood vessel growth (neovascularization) and increased vascular permeability, exacerbating retinal damage (Aiello et al., 1994).

In cell culture studies, hypoxia chambers have been used to mimic conditions with abnormally limited oxygen supply. A hypoxia chamber provides a controlled environment that allows the effects of specific levels of oxygen to be determined. Designed to fit inside existing laboratory incubators, this self-contained and sealed chamber helps to maintain a hypoxic environment with controlled oxygen levels and a stable temperature. Hypoxic conditions are achieved with specific gas mixtures, generally (1%, 5% or 10% oxygen; with 5% carbon dioxide and the balance nitrogen) (Wu and Yotnda, 2011). In general, oxygen concentrations of less than 2% are considered hypoxic, however, normoxic levels must be considered when selecting the experimental hypoxic conditions (Rinderknecht et al., 2021). An advantage of the use of a hypoxia chamber/incubator is that it is the most natural system to induce cellular hypoxic response, and it permits control of the oxygen fraction in the air surrounding the cultures within it (Rinderknecht et al., 2021). However, it has been shown that if the media is not pre- equilibrated against air containing the lower fraction of O2, it can take up to 24 hours for the average oxygen concentrations in the cell culture media to stabilize (Newby et al., 2005). It should also be noted that the partial pressure of O2 at the cell surface not only depends upon the fraction is surrounding air, but also the rate of diffusion to the cells, which typically depends upon the depth of the culture media (if not agitated) and the rate of oxygen consumption of the cells (Al-Ani et al., 2018).

In ocular studies, in addition to the hypoxia incubator/chamber, another commonly employed method for simulating hypoxia is using the hypoxia mimetic cobalt chloride (CoCl2). Compared to the hypoxia chamber, induction of hypoxia with the use of CoCl2 is simple and inexpensive, furthermore, it has the advantage of rapid induction of a cellular response that mimics hypoxia following treatment (Rinderknecht et al., 2021). However, this method has disadvantages, including possible toxicity and the fact that it does not fully mimic the cellular response to hypoxia, rather, it stabilizes hypoxia-inducible factor alpha proteins (HIF-1α and HIF-2α) (Rinderknecht et al., 2021). The precise mechanism by which CoCl2 stabilizes HIFs is not proven but is likely to involve replacement of ferrous iron(II) by Co2+ in the active site of HIF-prolyl hydroxylase enzymes (a.k.a. prolyl hydroxylase domain (PHD) proteins); thus, CoCl2 blocks proline hydroxylation of HIFs, which is the first step in their oxygen-induced polyubiquination and degradation by the proteosome (Muñoz-Sánchez and Chánez-Cárdenas, 2019). In addition, cobalt may inhibit the reduction of ferric iron (Fe3+) to ferrous iron (Fe2+) by ascorbic acid, thus inhibiting PHD activity; and/or Co2+ may bind directly to HIF alpha proteins and inhibit their ubiquination (Muñoz-Sánchez and Chánez-Cárdenas, 2019). Importantly, the set of genes induced by hypoxia and CoCl2 treatment may differ, as CoCl2 induction was reported to preferentially activate expression of HIF-1α-responsive genes while inhibiting the expression HIF-2α-responsive genes in hepatic cancer cells (Befani et al., 2013).

Here, we describe general protocols for inducing hypoxia in cell cultures using the hypoxia chamber and CoCl2. In addition, we describe a methodology for assessing protein levels of HIF-1α. Furthermore, we provide a summary of conditions applied to previous studies considering hypoxia chamber/incubator and CoCl2 in ocular cell cultures.

2. Materials and Supplies

2.1. Materials and Supplies for Hypoxia chamber Protocol

  • 2.1.1. Cell culture incubator

  • 2.1.2. Hypoxia chamber

  • 2.1.3. Pre-equilibrated cell culture media*

  • 2.1.4. Two (2) dishes with identical cell cultures prepared with previously equilibrated cell culture media: one will be used as control in the incubator and not in the hypoxia chamber (normoxia); and the other will be placed in the hypoxia chamber within the incubator

  • 2.1.5. Two (2) petri dishes (100 mm) containing 10 mL of sterile water each.

*Preparation: Pre-equilibrated cell culture media can be prepared in several ways: 1. By allowing the culture media to equilibrate under hypoxic conditions in the same hypoxic chamber until the media has reached the desired oxygen concentration, measured by a dissolved oxygen probe. 2. Alternatively, by allowing the culture media to equilibrate in hypoxic conditions in the same hypoxic chamber for at least 24 hours prior to experimental setup. 3. By bubbling nitrogen gas through culture medium, for 15 minutes (for an oxygen level of approximately 1.5%) to 30 minutes (for an oxygen level near 0%) (Newby et al., 2005).

2.2. Materials and Supplies for the use of CoCl2 in stabilizing Hypoxia-Inducible Factor-1α levels

  • 2.2.1. Cobalt (II) chloride hexahydrate, suitable for cell culture. Synonyms: Cobaltous chloride hexahydrate, Cobalt (II) chloride hexahydrate. Formula: CoCl2 · 6H2O. CAS Number: 7791-13-1. Molecular Weight: 237.93. Soluble in water (100 mg/mL) (Sigma-Aldrich)

  • 2.2.2. Cell culture media

  • 2.2.3. Cell culture incubator

  • 2.2.4. Prepare one culture without the use of CoCl2 to be used as a control.

2.3. Materials and Supplies for the detection of Hypoxia-Inducible Factor-1α (HIF-1α) as a marker of hypoxia

  • 2.3.1. 3-(N-Morpholino)propane sulfonic acid (MOPS)

  • 2.3.2. Ethylene glycol tetraacetic acid (EGTA)

  • 2.3.3. Ethylenediaminetetraacetic acid (EDTA)

  • 2.3.4. Triton x-100

  • 2.3.5. Protease inhibitor mini tablet; ThermoFisher Scientific, Cat #PIA32955

  • 2.3.6. 10x Tris Buffered Saline

  • 2.3.7. Tween-20

  • 2.3.8. Bovine Serum Albumin (BSA)

  • 2.3.9. Human/Mouse/Rat HIF-1 alpha/HIF-1A Antibody; (1:1000) R&D Systems, Cat #MAB15362.

  • 2.3.10. Anti-mouse IgG HRP-linked; (1:8000); GE healthcare Lifesciences, Cat# NA931

  • 2.3.11. Chemiluminescent substrate solution (suggested: SuperSignal West Dura, ThermoFisher)

3. Detailed Methods

3.1. Detailed methods for the use of the Hypoxia chamber

  • 3.1.1. Open the hypoxia chamber, remove lid and trays, and check integrity of the O-ring

  • 3.1.2. Add 2 petri dishes (100 mm) containing 10 mL of sterile water to the chamber base, for maintaining humidity in the chamber.

  • 3.1.3. Place the chamber tray in the chamber and ensure that trays are properly seated in the base.

  • 3.1.4. Place the cell culture dish containing cells into the chamber

  • 3.1.5. Place the lid on the chamber and secure by pushing down

  • 3.1.6. Close the chamber ring clamp, ensuring a hermetic closure of the chamber

  • 3.1.7. Connect the inlet port of the tubing to the hypoxic gas tank, containing the desired hypoxic gas mix (e.g., 1% oxygen, 5% carbon dioxide, balance nitrogen)

  • 3.1.8. Open both the inlet and outlet tubing clamps

  • 3.1.9. Open gas cylinder valve to flush the chamber

  • 3.1.10. Adjust the working output pressure gauge to 2 in mmHg pressure using the pressure adjuster knob, allow the gasflow chamber to completely purge the chamber for ≥8 minutes

  • 3.1.11. Turn off gas flow

  • 3.1.12. Quickly close tubing clamp 1

  • 3.1.13. Quickly close tubing clamp 2

  • 3.1.14. Disconnect the chamber from the gas tank.

Of note, normoxic levels must be considered when selecting the experimental hypoxic conditions. For ocular cells, refer to Table 1 for previously studied conditions.

Table 1.

Summary of studies employing low oxygen levels for evaluating optical cells.

Cell/tissue type Species % Oxygen Reference
661W cell line Mouse 0.2% (Kiessling et al., 2022)
661W cell line Mouse 0.5% (Tsui et al., 2013)
661W cell line Mouse 1% (Inoue et al., 2014; Kunimi et al., 2021; Kunimi et al., 2019; N Li et al., 2019; N Li et al., 2020; X Liu et al., 2020b; Shelby et al., 2015; Y Sun et al., 2021; Sweigard et al., 2015; Xu et al., 2019)
661W cell line Mouse 3% (Produit-Zengaffinen et al., 2016)
661W cell line Mouse 5% (J Liu et al., 2020a)
Astrocytes Human 1% (Mense et al., 2006)
Astrocytes Rat <0.7% (Watkins et al., 2013)
Fetal retinal pigment epithelial cells (hfRPE) Human 1% (H Wang et al., 2011)
Fetal retinal pigmented epithelial cells (RPE), F-0202 Human 1% (Udono et al., 2001)
induced pluripotent stem cell (iPSC)-derived RPE cells (iRPEs) iPSC-RPE Human 4% (Peters et al., 2022)
Müller cells Rat <0.7% (Watkins et al., 2013)
Müller cells Mouse 1% (N Li et al., 2019; N Li et al., 2020)
Müller glial cell (MGC) line MIO-M1 Human 0%# (Saint-Geniez et al., 2013)
Müller glial cell (MGC) line MIO-M1 Human 1% (Subirada et al., 2022; Y Sun et al., 2021)
Retina (cultured) Monkey 0%* (Nakajima et al., 2006)
Retina (cultured) Rat 0%* (Tamada et al., 2002)
Retinal endothelial cells (HRECs) Human 0.2% (Klee et al., 2020)
Retinal Ganglion Cell (RGC) - Primary Rat 5% (Chen et al., 2007; Yamagishi and Aihara, 2014; Yamagishi et al., 2011)
Retinal microvascular endothelial cells (RMEC) Rat <0.7% (Watkins et al., 2013)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 0%* (Xie et al., 2021; Zheng et al., 2016)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 0.2% (Klee et al., 2020)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human <0.25% (Zhou et al., 2018)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 0.5% (Harned et al., 2014; Sradhanjali et al., 2017; M Sun et al., 2022)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 1% (Arjamaa et al., 2017; Dougherty et al., 2008; Hwang et al., 2020; Kunimi et al., 2019; Shoda et al., 2020; Takei et al., 2017; Tang et al., 2022; Udono et al., 2001; Yoon et al., 2014; J Zhang et al., 2015; Zhu et al., 2022)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 2% (Golan et al., 2014)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 3% (Forooghian et al., 2007; Henning et al., 2022)
Retinal pigment epithelial (RPE) cell line D407 Human 1% (Feng et al., 2020; NN Liu et al., 2015; Tang et al., 2022; Udono et al., 2001)
Retinal pigment epithelial (RPE) cell line HRPEpiC Human 0.2% (Klee et al., 2020)
Retinal pigment epithelial (RPE) cell line hTERT RPE1 Human 1% (Menegakis et al., 2021; Yamamoto et al., 2021)
Retinal pigmented epithelial cells (RPE) Human 1% (Buczek-Thomas et al., 2019; Fuchshofer et al., 2009; Hollborn et al., 2018; Kernt et al., 2012; Ma et al., 2012; Rosen et al., 2015; P Zhang et al., 2009)
Retinal pigmented epithelial cells (RPE) Human 3% (Kurihara et al., 2016)
Retinal pigmented epithelial cells (RPE) Monkey ≤1% (Nakajima et al., 2017)
Retinal pigmented epithelial cells (RPE) Mouse 3% (Kurihara et al., 2016)
Retinal pigmented epithelial cells (RPE) Porcine 2% (Touhami et al., 2022)
Retinal pigmented epithelial cells (RPE) Rat <0.7% (Watkins et al., 2013)
Retinal Cell Line (R28) Rat 0.2% (Y Yang et al., 2022)
Retinoblastoma cell lines (Y79 and Weri-Rb1) Human 0.5% (Sradhanjali et al., 2017)
Retinoblastoma cell lines (Y79 and Weri-Rb1) Human 1% (Q Yang et al., 2017)
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*

Treatment described as 95% N2 and 5% CO2

#

Treatment described as complete deprivation of oxygen or anoxia

The pre-equilibration time required for the cell culture medium to reach the desired hypoxic oxygen concentration depends on both the volume of the medium and the target oxygen level. In this experimental protocol, we employ an initial 8-minute hypoxic air purge, followed by a 24-hour period for the pre-equilibration of the cell culture medium before commencing the experiments. Cells were cultured in 4 mL of medium, in 60 mm petri dishes.

Sampling should be performed as quickly as possible, and after sampling or manipulation, the chamber must be re-gassed to restore the desired hypoxic conditions. Allow the chamber to re-equilibrate before further experiments. Modifications to this protocol should be verified by confirming HIF-1α stabilization, as described in item 3.3. Protocol for the detection of Hypoxia-Inducible Factor-1α (HIF-1α) as a marker of hypoxia. It is crucial to maintain consistent hypoxic conditions throughout the cell culture experiments to obtain accurate and reproducible results.

3.2. Detailed methods for the use of CoCl2 in stabilizing Hypoxia Inducible Factor1-α levels

  • 3.2.1. Estimate the final cell culture volume needed

  • 3.2.2. Prepare a 23.793 mM stock solution immediately before use (5.6611 mg/mL) in sterile PBS

  • 3.2.3. Transfer the appropriate volume of stock solution directly to the complete cell culture media to obtain the desired concentration*, adjust according to final desired volume. Refer to table 2 for dilution examples

  • 3.2.4. Plate cells accordingly and transfer cells into regular cell culture incubator.

Table 2.

Examples of 23.793 mM CoCl2 stock solution dilutions into 10 mL of cell culture media.

Desired final CoCl2 concentration (μM) Stock Volume (μL) Stock concentration Final cell culture media volume
50 21.01 23.793 mM 10 mL
100 42.03
200 84.06
250 105.07
300 126.09
350 147.10
400 168.12
500 210.15
600 252.18
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Of note, when using CoCl2 for the first time, test a range of concentrations to establish non-toxic working concentrations for your cell type and experimental conditions, as this reagent has been demonstrated to reduce cell viability in concentrations as low as 300 μM (Fung et al., 2016; Kuehn et al., 2017; Rodriguez et al., 2021). For ocular cells, employed concentrations typically fall within the range of 50–600 μM. Refer to Table 3 for previously studied conditions in specific cell types.

Table 3.

Summary of studies employing cobalt chloride to induce hypoxia in optical cells.

Cell type Species Dose Reference
661W cell line Mouse 200 μM (Kunimi et al., 2019; Lee et al., 2020)
661W cell line Mouse 300 μM (Rodriguez et al., 2021)
Endothelial cells Human 150 μM (Jiang et al., 2021)
Müller cells Rabbit 250 mM (Lu et al., 2013)
Müller cells Rat 500 μM (X Zhang et al., 2012)
Müller glial cell line MIO-M1 Human 250 μM (Ahmad et al., 2021)
Müller glial cell line MIO-M1 Human 300 μM (Abu El-Asrar et al., 2021)
Müller glial cell line MIO-M1 Human 75 μM - 500 μM (Subirada et al., 2022)
Retina Mouse 200 μM (Y Wang et al., 2017)
Retina Porcine 300 μM (Mueller-Buehl et al., 2021 ; Tsai et al., 2020)
Retinal endothelial cells (HRECs) Human 200 μM (Long et al., 2019)
Retinal Ganglion Cell (RGC) - Primary Rat 100 μM (Youale et al., 2022)
Retinal microvascular endothelial cells (HRMECs) Human 300 μM (Abu El-Asrar et al., 2022)
Retinal Müller glial cells Human 300 μM (Abu El-Asrar et al., 2022)
Retinal pigmented epithelial cells (RPE) Human 100 μM (Ma et al., 2012)
Retinal pigmented epithelial cells (RPE) Human 100 – 350 μM (Alivand et al., 2016; Alivand et al., 2017)
Retinal pigmented epithelial cells (RPE) Human 150 μM (Hollborn et al., 2018; Rosen et al., 2015)
Retinal pigmented epithelial cells (RPE) Human 200 μM (ZX Zhang et al., 2011)
Retinal pigmented epithelial cells (RPE) Human 8 mM, 12 mM (Cheng et al., 2019)
Retinal pigmented epithelial cells (RPE) Mouse 200 μM (YQ Wang et al., 2010)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 10 – 1000 μM (Guerra et al., 2021)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 50 – 300 μM (Y Wang et al., 2016)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 100 μM (Du et al., 2013; Hwang et al., 2020; Sant et al., 2018)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 100 – 1000 μM (Chang et al., 2017)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 150 μM (Alzhrani et al., 2017; Lai et al., 2017; Veltmann et al., 2016)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 200 μM (Bahrami et al., 2019; Ibuki et al., 2020 ; Kunimi et al., 2019; Shoda et al., 2020; Takei et al., 2017; H Zhang et al., 2020; Y Zhang et al., 2018; Zhao et al., 2015; Zhu et al., 2016)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 200 – 800 μM (Gu et al., 2021)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 300 μM (Zheng et al., 2016)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 600 μM (Zhou et al., 2018)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 600 μM (KR Li et al., 2013)
Retinal pigment epithelial (RPE) cell line ARPE-19 Human 8 mM (Cheng et al., 2019)
Retinal pigment epithelial (RPE) cell line hTERT RPE1 Human 25 – 75 μg/mL (Qiao et al., 2021)
Retinal pigment epithelial (RPE) cell line hTERT RPE1 Human 75 μg/mL (Qiao et al., 2021)
Retinal Cell Line (R28) Human 0.5 mM (Thakur et al., 2021)
Retinoblastoma cell lines (Y79 and Weri-Rb1) Human 50 – 400 μM (Q Yang et al., 2017)
Retinoblastoma cell lines (Y79 and Weri-Rb1) Human 100 – 300 μM (Sradhanjali et al., 2017)
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3.3. Protocol for the detection of Hypoxia-Inducible Factor-1α (HIF-1α) as a marker of hypoxia

3.3.1. Solutions

3.3.1.1. Cell lysis buffer

  • Transfer 9.56 mL of Ultrapure water into a 15 mL falcon tube

  • Add 200 μL of 1M MOPS

  • Add 40 μL of 500 mM EGTA

  • Add 100 μL of 0.5 M EDTA

  • Add 100 μL of 10% Triton x-100

  • Add 1 Thermo Scientific protease inhibitor mini tablet (PIA32955)

  • Keep solution on ice during experimental use

  • This solution can be aliquoted and stored at −20°C

3.3.1.2. TBS-T

  • 100 mL 10x Tris Buffered Saline (TBS)

  • 900 mL ultrapure water

  • 1 mL Tween-20

3.3.1.3. Blocking buffer

  • Weigh 2.5 g of BSA (Bovine Serum Albumin) and transfer into appropriate 50 mL flask

  • Add TBS-T solution as described above for a final 50 mL volume

  • Mix well

  • Maintain refrigerated

3.3.2. Procedure

  • 3.3.2.1. Remove samples from incubator and quickly lyse cells in a solution containing 20 mM MOPS, 2 mM EGTA, 5 mM EDTA, 1% Triton-X-100, 1 mM DTT and protease inhibitors, maintaining samples on ice

  • 3.3.2.2. Centrifuge cell lysates at 16,000 × g in for 10 minutes at 4°C

  • 3.3.2.3. Measure protein concentration in supernatant using preferred methodology (suggested: RC DC Protein Assay Kit, Bio-Rad)

  • 3.3.2.4. Add appropriate volume of preferred loading dye. Keep at room temperature for 25–30 minutes. Suggested loading die: 2x Laemmli Sample Buffer (# 1610737)

  • 3.3.2.5. Load equal amounts of protein (suggested: 20 μg) from each sample into the wells of a 4% to 15%. SDS-PAGE gel and proceed with electrophoresis for protein separation

  • 3.3.2.6. Transfer the protein from gel to a polyvinylidene fluoride membrane

  • 3.3.2.7. Block membrane for 1 hour in Blocking buffer

  • 3.3.2.8. Incubate the membrane overnight with appropriate dilution of the primary antibody (HIF 1-α) in blocking solution. Suggested antibody: Human/Mouse/Rat HIF-1 alpha/HIF-1A Antibody; (1:1000) R&D Systems, Cat #MAB1536

  • 3.3.2.9. Wash the membrane three times for 5–10 minutes each in TBS-T solution

  • 3.3.2.10. Incubate the membrane with appropriate dilution (suggested 1:8000) of the secondary antibody in blocking solution. Suggested antibody: Anti-mouse IgG HRP-linked; (1:8000); GE healthcare Lifesciences, Cat# NA931

  • 3.3.2.11. Wash the membrane three times for 5–10 minutes each in TBS-T solution

  • 3.3.2.12. Add preferred chemiluminescent substrate solution (suggested: SuperSignal West Dura, ThermoFisher)

  • 3.3.2.13. Acquire image

4. Potential Pitfalls and Trouble Shooting

When hypoxia chamber is being used, cell culture media needs to be pre-equilibrated before the start of the experiment, as it may take up to 24 hours for the media to reach hypoxic levels (Newby et al., 2005). Variability in oxygenation levels at the beginning of the experiment may compromise the reproducibility of results. In addition, reoxygenation of cell cultures may occur immediately if the O-ring is compromised, and upon opening of the hypoxia chamber, thus, it is imperative that the chamber remains sealed throughout the duration of the study and opened only at the collection time. If a time-course study is being conducted, the use of additional chambers will be necessary. Ideally, an oxygen analyzer or indicator should be placed in the hypoxia chamber to monitor the maintenance of hypoxic conditions, such as Forensics Detectors, Model: FD-90A-O2. Sample collection should occur as quickly as possible, and samples should be denatured promptly, as oxygen sensing may continue to occur even in the cell lysates (Wenger et al., 2015).

If CoCl2 is used to stabilize hypoxia-inducible factor alpha proteins (HIF-1α and HIF-2α), a range of concentrations should be tested to establish an effective and non-toxic working concentration for your cell type (Rinderknecht et al., 2021). In addition, it is important to note that CoCl2 does not entirely mimic cellular response to hypoxia, thus, caution should be employed when interpreting the results.

Figure 1.

Figure 1.

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Hypoxia chamber parts (1) Chamber lid; (2) Ring clamp; (3) O-ring; (4) Chamber tray; (5) Petri dishes containing sterile water; (6) Inlet tubing with clamp; (7) Outlet tubing with clamp.

Figure 2.

Figure 2.

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Schematic protocol for the use of the hypoxia chamber. (1) Open the hypoxia chamber; (2) Add petri dishes containing of sterile water to the chamber base; (3) Identify the correct position of the chamber tray; (4) Add the chamber tray, making sure it is secured in place; (5) Add the cell cultureware containing one of the twin cultures in the chamber; (6) Add the chamber lid; (7) Close the chamber ring clamp, ensuring a hermetical closure of the chamber; (8) Connect the chamber to the hypoxic gas tank; (9) Open first tubing clamp; (10) Open second tubing clamp; (11) Open gas cylinder valve to flush the chamber; (12) Adjust the working output pressure gauge to 2 in.Hg pressure using the pressure adjuster knob, allowing the chamber to purge for 8 minutes; (13) Turn off gas flow; (14) Quickly close tubing clamp 1; (15) Quickly close tubing clamp 2; (16) Disconnect the chamber from the gas tank.

Figure 3.

Figure 3.

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Western blot of HIF-1α protein in mouse immortalized cone photoreceptor cell line 661W, after 24 hours of incubation in hypoxia chamber (5%Carbon dioxide, 1%Oxygen, balance Nitrogen).

Funding

D.N.Z and S.F.A supported by NIH R01EY020823. S.F.A also supported by NIH R01EY029349, NIH R01EY031961 and Research to Prevent Blindness (RPB). B.M.S supported by Training Grant T32AR07080 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases. The work was supported by an NEI Vision Core Research Grant NIH P30EY007003. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

Declaration of competing interest

The authors declare no conflict of interest.

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