Name: in vitro method to study sex-based differences in conjunctival goblet cells
Uploaded: 2023-07-28T14:50:00
Description: Watch this Scientific Journal Video about In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells at JoVE.com

The work is funded by the National Eye Institute Grant EY019470 (D.A.D).

All human tissue was donated to the eye bank with prior informed consent and authorization of the donor for use in scientific research. Use of the human conjunctival tissue was reviewed by the Massachusetts Eye and Ear Human Studies Committee and determined to be exempt and not meeting the definition of research with human subjects.
1. Primary human goblet cell culture
<ol>
	<li>From the eye bank, obtain human conjunctival tissue16.</li>
	<li>Prepare ...

<ol>
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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+negatively+regulates+epithelial+wound+healing+and+protective+lipid+mediator+circuits+in+the+cornea.">Estrogen negatively regulates epithelial wound healing and protective lipid mediator circuits in the cornea.</a> FASEB Journal. 26, 1506-1516 (2012).</li><li>Sullivan, D. A., Block, L., Pena, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+androgens+and+pituitary+hormones+on+the+structural+profile+and+secretory+activity+of+the+lacrimal+gland.">Influence of androgens and pituitary hormones on the structural profile and secretory activity of the lacrimal gland.</a> Acta Ophthalmologica Scandinavica. 74, 421-435 (1996).</li><li>Schaumberg, D. A., Dana, R., Buring, J. E., Sullivan, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+dry+eye+disease+among+US+men:+estimates+from+the+Physicians'+Health+Studies.">Prevalence of dry eye disease among US men: estimates from the Physicians' Health Studies.</a> Archives of Ophthalmology. 127, 763-768 (2009).</li><li>Tellefsen N&#248;land, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+and+age+differences+in+symptoms+and+signs+of+dry+eye+disease+in+a+Norwegian+cohort+of+patients.">Sex and age differences in symptoms and signs of dry eye disease in a Norwegian cohort of patients.</a> The Ocular Surface. 19, 68-73 (2021).</li><li>Sullivan, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TFOS+DEWS+II+Sex,+gender,+and+hormones+report.">TFOS DEWS II Sex, gender, and hormones report.</a> The Ocular Surface. 15, 284-333 (2017).</li><li>Meester, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeXY+chromosomes+and+the+immune+system:+reflections+after+a+comparative+study.">SeXY chromosomes and the immune system: reflections after a comparative study.</a> Biology of Sex Differences. 11, 3 (2020).</li><li>Yang, J. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone+replacement+therapy+reverses+the+decrease+in+natural+killer+cytotoxicity+but+does+not+reverse+the+decreases+in+the+T-cell+subpopulation+or+interferon-gamma+production+in+postmenopausal+women.">Hormone replacement therapy reverses the decrease in natural killer cytotoxicity but does not reverse the decreases in the T-cell subpopulation or interferon-gamma production in postmenopausal women.</a> Fertility and Sterility. 74, 261-267 (2000).</li><li>Orucoglu, F., Akman, M., Onal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+age,+refractive+error+and+gender+related+changes+of+the+cornea+and+the+anterior+segment+of+the+eye+with+Scheimpflug+imaging.">Analysis of age, refractive error and gender related changes of the cornea and the anterior segment of the eye with Scheimpflug imaging.</a> Contact Lens &amp; Anterior Eye. 38, 345-350 (2015).</li><li>Strobbe, E., Cellini, M., Barbaresi, U., Campos, E. 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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age,+gender,+and+interracial+variability+of+normal+lacrimal+gland+volume+using+MRI.">Age, gender, and interracial variability of normal lacrimal gland volume using MRI.</a> Ophthalmic Plastic and Reconstructive Surgery. 30, 388-391 (2014).</li><li>Sullivan, B. D., Evans, J. E., Dana, M. R., Sullivan, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+aging+on+the+polar+and+neutral+lipid+profiles+in+human+meibomian+gland+secretions.">Influence of aging on the polar and neutral lipid profiles in human meibomian gland secretions.</a> Archives of Ophthalmology. 124, 1286-1292 (2006).</li><li>Ebeigbe, J. A., Ebeigbe, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+sex+hormone+levels+on+tear+production+in+postmenopausal+Nigerian+women.">The influence of sex hormone levels on tear production in postmenopausal Nigerian women.</a> African Journal of Medicine and Medical Sciences. 43, 205-211 (2014).</li><li>Suzuki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen's+and+progesterone's+impact+on+gene+expression+in+the+mouse+lacrimal+gland.">Estrogen's and progesterone's impact on gene expression in the mouse lacrimal gland.</a> Investigative Ophthalmology &amp; Visual Science. 47, 158-168 (2006).</li><li>Shatos, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+cultured+human+conjunctival+goblet+cells.">Isolation and characterization of cultured human conjunctival goblet cells.</a> Investigative Ophthalmology &amp; Visual Science. 44, 2477-2486 (2003).</li><li>Huang, A. J., Tseng, S. C., Kenyon, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+rat+conjunctival+goblet+cells.">Morphogenesis of rat conjunctival goblet cells.</a> Investigative Ophthalmology &amp; Visual Science. 29, 969-975 (1988).</li><li>Li, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolvin+D1+and+aspirin-triggered+resolvin+D1+regulate+histamine-stimulated+conjunctival+goblet+cell+secretion.">Resolvin D1 and aspirin-triggered resolvin D1 regulate histamine-stimulated conjunctival goblet cell secretion.</a> Mucosal Immunology. 6, 1119-1130 (2013).</li><li>Dartt, D. A., Masli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjunctival+epithelial+and+goblet+cell+function+in+chronic+inflammation+and+ocular+allergic+inflammation.">Conjunctival epithelial and goblet cell function in chronic inflammation and ocular allergic inflammation.</a> Current Opinion in Allergy and Clinical Immunology. 14, 464-470 (2014).</li><li>Mantelli, F., Arg&#252;eso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+ocular+surface+mucins+in+health+and+disease.">Functions of ocular surface mucins in health and disease.</a> Current Opinion in Allergy and Clinical Immunology. 8, 477-483 (2008).</li><li>Garc&#237;a-Posadas, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+functional+performance+of+a+commercial+human+conjunctival+epithelial+cell+line.">Characterization and functional performance of a commercial human conjunctival epithelial cell line.</a> Experimental Eye Research. 223, 109220 (2022).</li><li>Shatos, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+characterization,+and+propagation+of+rat+conjunctival+goblet+cells+in+vitro.">Isolation, characterization, and propagation of rat conjunctival goblet cells in vitro.</a> Investigative Ophthalmology &amp; Visual Science. 42, 1455-1464 (2001).</li><li>Welshons, W. V., Wolf, M. F., Murphy, C. S., Jordan, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogenic+activity+of+phenol+red.">Estrogenic activity of phenol red.</a> Molecular and Cellular Endocrinology. 57, 169-178 (1988).</li><li>Berthois, Y., Katzenellenbogen, J. A., Katzenellenbogen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenol+red+in+tissue+culture+media+is+a+weak+estrogen:+implications+concerning+the+study+of+estrogen-responsive+cells+in+culture.">Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture.</a> Proceedings of the National Academy of Sciences of the United States of America. 83, 2496-2500 (1986).</li><li>Garc&#237;a-Posadas, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+IFN-&#947;+with+cholinergic+agonists+to+modulate+rat+and+human+goblet+cell+function.">Interaction of IFN-&#947; with cholinergic agonists to modulate rat and human goblet cell function.</a> Mucosal Immunology. 9, 206-217 (2016).</li><li>Li, D., Jiao, J., Shatos, M. A., Hodges, R. R., Dartt, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+VIP+on+intracellular+[Ca2+],+extracellular+regulated+kinase+1/2,+and+secretion+in+cultured+rat+conjunctival+goblet+cells.">Effect of VIP on intracellular [Ca2 ], extracellular regulated kinase 1/2, and secretion in cultured rat conjunctival goblet cells.</a> Investigative Opthalmology &amp; Visual Science. 54, 2872-2884 (2013).</li><li>Contr&#242;, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+steroid+hormone+receptors,+their+ligands,+and+nuclear+and+non-nuclear+pathways.">Sex steroid hormone receptors, their ligands, and nuclear and non-nuclear pathways.</a> AIMS Molecular Science. 2, 294-310 (2015).</li><li>Valley, C. C., Solodin, N. M., Powers, G. L., Ellison, S. J., Alarid, E. 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A., Gorski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anomalous+behavior+of+protein+synthesis+inhibitors+on+the+turnover+of+the+estrogen+receptor+as+measured+by+density+labeling.">Anomalous behavior of protein synthesis inhibitors on the turnover of the estrogen receptor as measured by density labeling.</a> Endocrinology. 119, 1454-1461 (1986).</li><li>Yang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-based+differences+in+conjunctival+goblet+cell+responses+to+pro-inflammatory+and+pro-resolving+mediators.">Sex-based differences in conjunctival goblet cell responses to pro-inflammatory and pro-resolving mediators.</a> Scientific Reports. 12, 16305 (2022).</li></ol>

Investigating the sex-based differences in ocular tissues helps understand the processes of diseases, especially dry eye and allergic conjunctivitis, which disproportionately affect one sex4,5,6. Even though animal models can be used for these studies, data obtained directly from human tissue are essential due to the highest similarity to the human cells in vivo. The conjunctival tissues used in the current experimental...

Sex-based differences affect multiple processes of the ocular surface1,2,3. The clinical manifestation of these sex-based differences is the difference in the prevalence of many ocular surface diseases between men and women, such as dry eye and conjunctivitis4,5,6. Evidence suggests that sex-based differences arise from multiple biological levels, including the different profiles of genes on X and Y chromosomes7 and the effects of hormones8. Studying the molecular basis of sex-based differences can provide a better understanding of disease and, eventually, improve personalized medicine.
The ocular surface comprises the overlying tear film, cornea, and conjunctiva. Sex-based differences are observed in multiple components of the ocular surface, including the tear film9,10, cornea11, the lacrimal gland12,13, and meibomian glands that also secrete tears12. Numerous mechanistic studies have investigated the effect of sex hormones on the cornea and its associated components14,15; however, little is known about the sex-based differences in the conjunctiva and its goblet cells. The conjunctiva is a mucous membrane that covers the sclera and the inner surface of the eyelid. The epithelium of the conjunctiva is comprised of nonkeratinizing, multi-layered, stratified squamous cells16.
Among the stratified squamous cells of the conjunctiva, there are goblet cells (CGCs) interspersed at the apical surface of the epithelium. These goblet cells are characterized by the large number of secretory granules located at the apical pole17. CGCs synthesize and secrete the gel-forming mucin MUC5AC to moisturize the ocular surface and lubricate it during blinking17. Mucin secretion is tightly regulated by the intracellular [Ca2+] ([Ca2+]i) and the activation of the Ras-dependent extracellular signal-regulated kinase (ERK1/2)18. Inability to secrete mucin results in dryness of the ocular surface and sequelae of pathological abnormalities. On an inflamed ocular surface, however, extensive mucin secretion stimulated by inflammatory mediators leads to a perception of stickiness and itchiness of the eye19. These conditions with disturbed mucin secretion will eventually lead to deterioration of the ocular surface.
The role of goblet cells as the major source of ocular mucin has long been recognized20, however, the sex-based differences in mucin regulation in both physiological and pathological states remain undiscovered. An in vitro system would be useful to monitor the function of goblet cells without the hormonal effect or with a precisely controlled level of sex hormones. Even though a conjunctival epithelial cell line has developed21, there is no goblet cell line with functional mucin secretion available. Therefore, we modified our developed primary human CGC culture to establish a method to analyze the sex-based difference in vitro16, and present it as below.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% trypsin with 1x EDTA</td>
			<td>Gibco (Grand Island, NY)</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid</td>
			<td>Fisher Bioreagent (Pittsburgh, PA)</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced RPMI media</td>
			<td>Gibco (Grand Island, NY)</td>
			<td>12633020</td>
			<td></td>
		</tr>
		<tr>
			<td>carbachol</td>
			<td>Cayman Chemical (Ann Arbor, MI)</td>
			<td>144.86</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovin Serum</td>
			<td>R&#38;D (Minneapolis, MN)</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2- acetoxymethyl ester&#160;</td>
			<td>Thermo Fisher Scientific (Waltham, MA, USA)</td>
			<td>F1221</td>
			<td></td>
		</tr>
		<tr>
			<td>Human conjunctival tissue</td>
			<td>Eversight Eye Bank (Ann Arbor, MI)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>inorganic salt for KRB buffer</td>
			<td>Sigma-Aldrich (St. Louis, MO)</td>
			<td></td>
			<td>Any brand will work</td>
		</tr>
		<tr>
			<td>L-glutamine&#160;</td>
			<td>Lonza Group (Basel, Switzerland)</td>
			<td>17-605F</td>
			<td></td>
		</tr>
		<tr>
			<td>non-essential amino acids</td>
			<td>Gibco (Grand Island, NY)</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Gibco (Grand Island, NY)</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>phenol red-free RPMI media&#160;</td>
			<td>Gibco (Grand Island, NY)</td>
			<td>11835055</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic acid F127</td>
			<td>MilliporeSigma (Burlington, MA, USA)</td>
			<td>P2443-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 culture medium</td>
			<td>Gibco (Grand Island, NY)</td>
			<td>21875034</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpel</td>
			<td>Thermo Fisher Scientific (Waltham, MA, USA)</td>
			<td>12460451</td>
			<td>Any sterile surgical scalpel can work</td>
		</tr>
		<tr>
			<td>sodium pyruvate</td>
			<td>Gibco (Grand Island, NY)</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>sulfinpyrazone</td>
			<td>MilliporeSigma (Burlington, MA, USA)</td>
			<td>S9509-5G</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro method to study sex-based differences in conjunctival goblet cells

Dry eye is a multi-factorial disease affecting ocular surface health, with a profoundly higher prevalence in women. Disruption of the gel-forming mucin that is secreted by conjunctival goblet cells (CGCs) onto the ocular surface contributes to multiple ocular surface diseases. The elimination of exogenous sex hormones is essential to obtain consistent results during in vitro study of sex-based differences in CGCs. This paper describes a method to minimize the presence of exogenous hormones in the study of sex-based differences in CGCs while maintaining their physiological function. CGCs from postmortem human donors of both sexes were cultured from pieces of the conjunctiva in RPMI medium with 10% fetal bovine serum (FBS) (referred to as the complete medium) until confluency. Nearly 48 h before the start of the experiments, CGCs were transferred to RPMI medium without phenol red or FBS but with 1% BSA (referred to as phenol-red-free medium). The normal cellular function was studied by measuring the increase in intracellular [Ca2+] ([Ca2+]i) after carbachol (Cch, 1 x 10-4 M) stimulation using fura 2/acetoxymethyl (AM) microscopy. The result shows that CGCs maintained normal function in the phenol-red-free media after 48 h. No significant difference in [Ca2+]i response was observed between phenol red-free RPMI medium and complete medium upon Cch stimulation. Therefore, we recommend using the phenol-red free RPMI medium with 1% BSA to eliminate exogenous hormones without altering the normal function of CGCs in the study of sex-based differences.

Human CGCs in primary culture grow to 80% confluency in approximately 14 days. The cell type was confirmed by immunofluorescence staining with antibodies to the goblet cell markers CK7 and HPA-125 (Figure 1). Even though the removal of FBS from the medium can eliminate the sex hormones, the lack of FBS could potentially affect the cellular response. To verify the hormone elimination method, a cholinergic agonist (carbachol, Cch 1 &#215; 10-4 M) was used as ...

Watch this Scientific Journal Video about In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells at JoVE.com

In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells

Phenol red-free/fetal bovine serum-free medium is a better option than advanced RPMI to eliminate exogenous hormones without altering the normal function of conjunctival goblet cells in the study of sex-based differences.

In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells

Dry eye is a multi-factorial disease affecting ocular surface health, with a profoundly higher prevalence in women. Disruption of the gel-forming mucin ...

By studying the goblet cell function in primary culture, we gain valuable insights into the mechanisms behind the sex-based differences in ocular surface health. We have modified the already-established conjunctival goblet cell primary culture method by eliminating hormones and estrogenic substances from the system, enabling more precise control of sex hormone levels. This modification enhances the accuracy and reliability of our findings.The intracellular calcium measurement is a method to study the goblet cell function under various stimuli and the intracellular signaling pathways triggered by each stimulus. The technique can be extended to drug development for ocular surface diseases like ocular allergy, dry eye disease, or conjunctivitis. This technique facilitates the screening of various molecule's effect on goblet cells under specific hormone levels.People new to this technique often struggle with separating conjunctival tissue from connective tissue. Our advice is to pay close attention to the texture and material properties of the tissue. Conjunctiva should be elastic and semi-transparent while the connective tissue is usually lighter in color and cotton fiber-like.To begin, using a sterile scalpel, mince the human conjunctival tissue into one millimeter cubed pieces. In a six-well plate, seed four pieces in each well containing one millimeter of complete RPMI medium. Make sure to anchor the pieces to the plate.Incubate the tissue pieces at 37 degrees Celsius, 5%carbon dioxide, and 95%air. Refresh the culture medium every second day. After 72 hours from seeding, remove the tissue plug and continue incubation until goblet cells reach 70 to 80%confluency.Next, rinse the cells with PBS and add 0.05%Trypsin EDTA to detach the cells from the bottom of the plate. Observe the cell detachment under a microscope. And once the cells detach, add a complete RPMI medium to deactivate the trypsin.Centrifuge the cell suspension at 150 G for five minutes and resuspend the pellet in phenol red-free RPMI medium. Then reseed the cell suspension onto a glass bottom culture dish for intracellular calcium concentration measurement. For Fura-2 AM loading, add Krebs-Ringer bicarbonate buffer containing 0.5%HEPES, 0.5%BSA, and 0.5 micromolar Fura-2 AM, eight micromolar pluronic acid F127, and 250 micromolar sulfinpyrazone into the glass bottom dish containing goblet cells.Incubate the cells for one hour at 37 degrees Celsius, protecting them from light. After incubation, wash the cells with Krebs-Ringer bicarbonate buffer containing 250 micromolar sulfinpyrazone. For intracellular calcium concentration measurement, place the dish containing cells loaded with Fura-2 under the microscope.Then add 200 times magnification. Locate a representative field containing 20 to 50 cells. Using the freehand function, draw an outline around each cell to distinguish it from the background fluorescence and wait for the software to automatically subtract background fluorescence from the measurement.Click the Start Experiment button and wait for eight to 15 seconds to determine the baseline calcium levels in the cells. Then carefully add the cholinergic agonist carbachol and continue measuring for at least 120 seconds or until calcium levels have returned to baseline. Using the change in peak intracellular calcium concentration, calculate the mean basal calcium level in each cell from the first eight to 15 seconds of measurements.Remove cells having a mean basal calcium level equal to or above 500 nanomolar from the dataset. Next, calculate the maximum intracellular calcium concentration for each cell. Then subtract the basal calcium level value from the maximum measured intracellular concentration for the same cell.Finally, average the change in peak intracellular calcium concentration for all the cells in a dish. The purity of human conjunctival goblet cells was confirmed via immunofluorescent staining, utilizing antibodies specific to the goblet cell markers, cytokeratin and Helix pomatia lectin 1. The results of the Fura-2 AM assay showed no significant differences between cells incubated in phenol red-free RPMI with 1%BSA and complete RPMI medium.However, a significant increase intracellular calcium response to carbachol was observed when comparing the advanced RPMI medium group to the complete RPMI medium group. The most important thing is to obtain primary human goblet cell culture of sufficient cell density. During growth, ensure that the tissue pieces stick to the culture plate for effective outgrowth.The researchers could explore the involvement of specific pathways by applying various inhibitors, targeting receptors or signaling molecules to the cells before the stimulation. The other parameters indicative of goblet cell function, like mucin secretion and deactivation of mitogen-activated protein kinases can also be assessed. The complimentary measurements provide a comprehensive understanding of the goblet cells'response to different stimuli.

in-vitro-method-to-study-sex-based-differences-conjunctival-goblet

Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Assessing Differences in Sperm Competitive Ability in Drosophila

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1. Sperm competition represents the competition between males after copulating with the same female 2, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males 4. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5.
Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9, which is assumed to be reflective of different competence attributes.
Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.

Assessing Differences in Sperm Competitive Ability in Drosophila

Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male.

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...