The authors received no funding for this work.

1. Culture of pHCECs and iHCECs
<ol>
	<li>Grow the pHCECs and iHCECs in human ocular epithelial medium (HOEM) with the following supplements: 6 mM L-glutamine, 0.002% cell media supplement O (Table of Materials), 1.0 &#181;M epinephrine, 0.4% cell media supplement P (Table of Materials), 5 &#181;g/mL rh insulin, 5 &#181;g/mL apo-transferrin, and 100 ng/mL hydrocortisone hemisuccinate in collagen-1 coated culture flasks (18 mL in a 75 cm2 culture flask).</li>
	<li>Change the medium in the flasks every 2-3 days by removing the medium after the cells grow to ~80% confluence. Add dissociation solution without phenol red and incubate the flask at 37 &#176;C until the cells are fully detached. Neutralize the dissociation solution with DMEM/F12 with serum and then centrifuge the cells at 500 &#215; g. Remove the supernatant medium and resuspend the cells in HOEM medium.</li>
</ol>
2. Determination of cell size using confocal microscopy
<ol>
	<li>Seed both pHCECs and iHCECs onto collagen-coated Petri dishes with glass-bottom coverslips at a concentration of 1 &#215; 105 with 1 mL of HOEM. Grow pHCECs and iHCECs, one set of cultures for 24 hours and another set of cultures for 48 h, in a 37 &#176;C incubator with 5% CO2.</li>
	<li>After the incubation period, stain the cells with 500 &#181;L of annexin staining buffer solution containing calcein (4 &#181;M), ethidium homodimer-1 (8 &#181;M), and annexin V (5 &#181;L in 500 &#181;L buffer-Alexa Fluor 647 conjugate) for 20 min at 37 &#176;C. After staining the cells, adjust the microscope for capturing the excitation/emission wavelengths of 488/515 nm for calcein-AM, 543/600 nm for ethidium homodimer-1, and 633/665 nm for annexin V using a confocal laser scanning microscope.</li>
	<li>Obtain the images and take Z-stacks to assess cell size in three dimensions.
	<ol>
		<li>To acquire the two-dimensional (2D) and three-dimensional (3D) images, find the area to image with an apochromat 40x/1.2 water objective. With the Argon (488) (first scan), HeNe1 (543) (second scan, and HeNe2 (633) (third scan) lasers, scan in three separate channels by using the beam splitters, HFT 488/543/633, NFT 635 Vis, and NFT 545 LP560, LP505.</li>
		<li>Use multi-track sequential scanning to reduce crosstalk. 
		NOTE: LP505 is used for channel 2 with the 488 laser to capture the emission of the calcein dye. LP560 is used in channel 3 with the 543 laser to capture the emission of the ethidium homodimer 1. NFT 635 is used with the 633 laser to capture the emission of the annexin V. The purpose of the HFT is to separate the excitation and emission light. NFT splits light by reflecting light &#60; specified wavelength to a separate channel while letting light &#62; a specified wavelength through to a second channel. LP filters block shorter wavelengths than the specified wavelength and let the longer wavelengths through.</li>
		<li>To image in 3D, set the confocal to Z-stack and scan at least 20 frames.</li>
	</ol>
	</li>
</ol>
3. Exposure of cells to UV radiation
<ol>
	<li>Seed the cells at 5 &#215; 104/mL of HOEM in each well of a 24-well collagen-1 coated culture plate and incubate at 37 &#176;C with 5% CO2 for 3 h. After the incubation period, reduce the volume of the medium in the wells to 300 &#181;L. Next, expose the cells to UV radiation (both UVA at 6.48 W/m2 and UVB at 1.82 W/m2 as both UVA and UVB tubes are turned on in the incubator at the same time) in a 37 &#176;C incubator for 5 and 20 min in separate experiments.</li>
	<li>After the UV radiation, add 200 &#181;L of fresh HOEM to each well and incubate for 20 h at 37 &#176;C with 5% CO2.</li>
	<li>After incubation, collect the cell supernatant in each well and transfer it into sterile 2 mL polypropylene tubes. Freeze at -80 &#176;C. To determine the cytokine levels released by the pHCECs and iHCECs after UV exposure, use a multiplex cytokine assay and follow the kit&#39;s instructions to quantify the following four cytokines: IL-6, IL-8, IL-1&#946;, and TNF-&#945;.</li>
	<li>Add metabolic assay reagent to the cells in the wells.</li>
	<li>Prepare 10% metabolic assay reagent in DMEM/F12. Replace the culture medium in each well with 1 mL of the 10% metabolic assay solution and incubate at 37 &#176;C with 5% CO2 for 4 h.</li>
	<li>Measure the fluorescence of each solution using a fluorescent plate reader at 530/590 nm excitation/emission wavelengths.</li>
</ol>
4. Exposure of cells to chemical toxins
<ol>
	<li>Seed cells at 5 &#215; 104/mL of HOEM in each well of a 24-well collagen-1 coated culture plate and incubate at 37 &#176;C with 5% CO2 for 3 h. After the incubation period, remove the medium and expose the cells to 1 mL of chemical toxins (BAK 0.001%, H2O2 0.01%, and SDS 0.0025% in phosphate-buffered saline (PBS)) for 5 and 15 min.</li>
	<li>After exposure to the chemical toxins, remove the toxins from the wells, rinse with 1 mL of PBS, and add 1 mL of HOEM to each well. After 20 h of incubation, perform a metabolic assay by replacing the medium with 1 mL of a 10% metabolic assay solution and incubating at 37 &#176;C with 5% CO2 for 4 h. Measure the fluorescence using a fluorescence multi-well plate reader at 530/590 nm excitation/emission wavelengths.</li>
	<li>Transfer the cell supernatants from the wells following the 20 h incubation into separate sterile 2 mL polypropylene tubes and freeze at -80 &#176;C. Quantify cytokines released by the pHCECs and iHCECs after exposure to the chemicals. Use the same multiplex platform used to assess the cytokines from the UV-treated cells. Use a multiplex cytokine assay following the kit&#39;s instructions to quantify the following four cytokines: IL-6, IL-8, IL-1&#946;, and TNF-&#945;.</li>
</ol>
5. Examination of cells exposed to various concentrations of BAK
<ol>
	<li>Seed cells at 1 &#215; 105/mL of HOEM in each well of a 24-well collagen-1 coated culture plate and incubate at 37 &#176;C with 5% CO2 for 24 h. After the incubation period, remove the medium and expose the cells to 1 mL of chemical toxins (BAK 0.001%, BAK 0.005%, and BAK 0.01% in PBS) for 5 min.</li>
	<li>After exposure, remove the chemical toxins, rinse the wells with 1 mL of PBS, and add 1 mL of HOEM to each well. After 20 h of incubation, stain the cells with 500 &#181;L of annexin staining buffer solution containing calcein (4 &#181;M), ethidium homodimer-1 (8 &#181;M), and annexin V (5 &#181;L in 500 &#181;L buffer-Alexa Fluor 647 conjugate) for 20 min at 37 &#176;C. Adjust the microscope to measure intensities of annexin V, calcein AM, and ethidium-1 staining at excitation/emission wavelengths of 630/675 nm, 495/515 nm, and 528/617 nm, respectively. Image the cells using the fluorescence microscope</li>
</ol>
6. Data analysis
<ol>
	<li>Carry out a normality test and a test for equal variances before performing an analysis of variance (ANOVA), Welch ANOVA, or Kruskal-Wallis test. Use the appropriate post-hoc test for comparing each group tested. Set p &#60; 0.05.</li>
</ol>

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The author, Lyndon W. Jones, over the past 3 years, through CORE, has received research support or lectureship honoraria from the following companies: Alcon, Allergan, Allied Innovations, Aurinia Pharma, BHVI, CooperVision, GL Chemtec,i-Med Pharma, J&amp;J Vision, Lubris, Menicon, Nature's Way, Novartis, Ophtecs, Ote Pharma, PS Therapy, Santen, Shire, SightGlass SightSage, and Visioneering. Lyndon Jones is also a consultant and/or serves on an advisory board for Alcon, CooperVision, J&amp;J Vision, Novartis, and Ophtecs. The other authors have nothing to disclose.

Potential differences in the use of two types of HCECs were assessed. Cells were placed in the same medium (HOEM) at identical concentrations of cells and then exposed to short and long periods of UV radiation and three ocular toxins. Doses of UV radiation and chemicals were selected based on their physiological effects, which were damaging enough to the cells to produce intermediate responses that could be compared. Exposure times of 5 and 20 min for UV radiation and 5 and 15 min for the selected doses of BAK (0.001%), ...

In vitro toxicology studies are performed to predict the toxic effects of chemicals and other agents that can cause damage to cells. In the assessment of toxicity to the cornea, human corneal epithelial cells (HCECs) have been used in models for evaluating these effects1,2,3,4. These models typically evaluate physiological effects such as changes to the cell&#39;s metabolic activity, cell proliferation, and other cell functions such as the production and release of inflammatory cytokines. For these toxicology studies, cells from various sources have been selected to assess the damaging effects of chemicals and UV radiation on HCECs2,3. pHCEC lines are available from companies that provide these cells from donor tissues of adults. Primary cells can be treated with dispase and gently scraped off the cornea for culture5. The cells are then tested for viruses and contamination and then shipped cryopreserved in 10% dimethyl sulfoxide.
The advantage of primary cell lines is that the cells are genetically identical to the cells of the donor. This is ideal, as an in vitro model should mimic the in vivo tissue as much as possible. The disadvantage of primary cell lines is that they have a limited number of cell divisions or passages6. The limited number of cells available restricts the number of experiments that can be conducted with a single primary culture, increasing the cost of the experiments.
Immortalized cell lines have also been used in cell culture toxicity models. However, unlike the primary cell line obtained from in vivo tissue, the immortalized cell line has been genetically altered. Immortalized cells are created by incorporating the genes of a virus into the DNA of primary cells6,7,8. Cells with successful viral gene incorporation are selected for the immortalized cell line. The advantage of immortalization is that it allows for indefinite rapid proliferation, providing an unlimited number of cells to perform multiple experiments using the same cell line. This allows for consistency between experiments and reduces the cost.
In addition to changes in the genes that limited cell proliferation, changes in the expression of genes of critical functionality could also occur9. Therefore, the disadvantage of using immortalized cells is that they may no longer represent the original in vivo cells in terms of their response to various external stimuli10. Comparisons have involved observing the toxic effects of chemicals on primary and immortalized human corneal keratocytes11, as well as immortalized HCECs and rabbit corneal epithelial primary cells12. The comparison between the effects of toxins on primary human keratocytes and immortalized keratocytes showed no significant differences11. Using the methods detailed in this article, the effectiveness of these assays to assess the toxicity of UV radiation and ocular toxins on pHCECs and iHCECs will be determined.
Three ocular toxins commonly used in in vitro assays were selected: BAK, H2O2, and SDS. BAK is a cationic preservative commonly used in ophthalmic solutions13,14, H2O2 is commonly used to disinfect contact lenses15, and SDS is an anionic surfactant found in detergents and shampoos14. Similar to ocular toxins, UV radiation can also cause significant damage to HCECs3. In addition, overexposure to UV can cause an ocular condition known as photokeratitis characterized by symptoms of tearing, light sensitivity, and a feeling of grittiness16.
There is an unlimited number of primary cell cultures that can be used and various immortalized cell lines that have been developed. Therefore, an investigation was undertaken to compare primary HCECs to an immortalized HCEC line to determine similarities and differences between models that incorporate these types of cells.
This investigation used microscopy to assess possible differences between pHCECs and iHCECs on cell physiological response to UV and toxins. The effects of UV radiation and chemicals on cell metabolic activity and inflammatory cytokine release for the two cell lines were also evaluated. The importance of determining the differences among the two cell lines is to understand the optimal use of these cell lines for evaluating: 1) the effect of UV radiation on cells, 2) the effects of toxins on cells, and 3) the resulting changes to metabolism, cell viability, and cell cytokine release for future studies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>75 cm2 Vented Flask</td>
			<td>Corning</td>
			<td>354485</td>
			<td>This is the BioCoat brand, collagen-coated</td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>costar</td>
			<td>3370</td>
			<td></td>
		</tr>
		<tr>
			<td>alamarBlue</td>
			<td>Fisher Scientific</td>
			<td>dal 1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin Staining buffer solution</td>
			<td>Invitrogen, Burlington, ON, Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin V</td>
			<td>Invitrogen, Burlington, ON, Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovert 100 microscope with a Zeiss confocal laser scanning microscope 510 system</td>
			<td>Carl Zeiss Inc., Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 48 Well plates</td>
			<td>Corning</td>
			<td>354505</td>
			<td>This is the BioCoat brand, collagen-coated</td>
		</tr>
		<tr>
			<td>Cytation 5</td>
			<td>BioTek</td>
			<td>CYT5MPV</td>
			<td>Can read fluorescence from 280 - 700 nm (for assay 540/590)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hycone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>glass bottom coverslips</td>
			<td>MatTek Corporation, Ashland, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human Corneal Epithelial Cells</td>
			<td>University of Ottawa</td>
			<td>N/A</td>
			<td>SV40-immortalized human corneal epithelial cells from Dr. M. Griffith (Ottawa Eye Research Institute, Ottawa, Canada) and have been characterized Griffith M, Osborne R, Munger R, Xiong X, Doillon CJ, Laycock NL, Hakim M, Song Y, Watsky MA. Functional human corneal equivalents constructed from cell lines. Science. 1999;286:2169-72.</td>
		</tr>
		<tr>
			<td>Human Ocular Epithelia Media (HOEM) with the following supplements:</td>
			<td>Millipore, Billerica, MA, USA</td>
			<td>SCMC001</td>
			<td>Epigro base media supplemented with 6 mM L-Glutamine, 0.002% EpiFactor O (cell media supplement O in article) , 1.0 &#956;M Epinephrine, 0.4% EpiFactor P (cell media supplement P in article), 5 &#956;g/mL rh Insulin, 5 &#956;g/mL Apo- Transferrin, and 100 ng/mL Hydrocortisone Hemisuccinate in Collagen-1 coated culture flasks (BioCoat, Corning, Tewksbury, MA, USA).</td>
		</tr>
		<tr>
			<td>Live Dead calcien and ethidium homodimer</td>
			<td>Invitrogen, Burlington, ON, Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MesoScale Discovery (MSD) QuickPlex SQ 120 instrument</td>
			<td>Rockville, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MSD Human Proinflammatory Panel II (4-Plex) V-Plex assay</td>
			<td>Rockville, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100x concentration so add 1 mL to each 99 mL of media</td>
		</tr>
		<tr>
			<td>Primary Corneal Epithelial Cells</td>
			<td>Millipore, Billerica, MA, USA</td>
			<td>SCCE016</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax fluorescence multi-well plate reader</td>
			<td>Molecular Devices, Sunnyvale, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express (cell disassociation solution)</td>
			<td>Fisher Scientific</td>
			<td>12605036</td>
			<td></td>
		</tr>
	</tbody>
</table>

determining the toxicity of uv radiation and chemicals on primary and immortalized human corneal epithelial cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

Cell size 
The primary and immortalized HCECs were visualized with three fluorescent dyes, which reflect three different stages of cell viability. Live cells are green (calcein-AM), dead cells are red (ethidium homodimer-1), and apoptotic cells are yellow (annexin V-computer-adjusted color for better visualization of the fluorescence signal). Live cells contain esterases in the cell cytoplasm and convert calcein-AM to calcein. Dead cells have cell membranes that are permeable to ethidium homodimer-1...

Watch this Scientific Journal Video about Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells at JoVE.com

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

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2.0K Views.  University of Waterloo. This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line. 

Video: Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...