The authors would like to thank Nandini Nallappan and Sharon Yeo for helping with the technical steps. The study was funded by a Start-Up Grant to KGC from Lee Kong Chian School of Medicine, Nanyang Technological University and by a Senior Clinician Scientist Award from the Singapore Ministry of Health's National Medical Research Council (NMRC) to LT (NMRC/ CSA/ 045/ 2012) and by a grant from the NMRC and administered by the National Health Innovation Centre to KGC and LT (NHIC-12D-1409007).

All ocular samples used in this study were collected from the Dry Eye Clinic at the Singapore National Eye Centre approved by SingHealth Centralized Institutional Review Board and the Nanyang Technological University Institutional Review Board, Singapore.
NOTE: Subjects underwent clinical tests to assess the extent of inflammation and the severity of tear dysfunction before IC was performed. Clinical tests included non-invasive tear break-up time (NI-TBUT)14 and conjunctival redness (hyperemia)15,16 assessed with a diagnostic instrument, Schirmer&#39;s test17, Standard Patient Evaluation of Eye Dryness (SPEED) questionnaire18, and corneal staining19.
1. Collection of Ocular Samples by IC
<ol>
	<li>After determination of the clinical status, anesthetize the participant&#39;s eyes by applying 1 - 2 drops of topical anesthesia, proparacaine hydrochloride, to the superior bulbar conjunctiva and inferior fornix. Wait for 4 - 5 min for the stinging sensation of the anesthetic to wear off.</li>
	<li>Collect samples from both nasal and temporal bulbar conjunctiva with two impression cytology devices. 
	NOTE: One device is used for the collection of two impression samples. Here, two devices were used for the collection of four impression membranes.
	<ol>
		<li>Position the impression cytology device tangentially on the conjunctiva. Press the push-button gently and hold for 2 - 3 s. 
		NOTE: The device comes with the membrane. Pressure is released automatically after pushing the button. It takes only few seconds to release the pressure and remove the device.</li>
	</ol>
	</li>
	<li>Release the membrane into a microcentrifuge tube containing 1 mL of culture media (Roswell Park Memorial Institute medium (RPMI) + 10% fetal bovine serum (FBS) + 1% Penicillin-Streptomycin (Pen Strep)) by a scalpel.</li>
	<li>Isolate cells from the membrane of the device by continuous scraping for around 1 min with a 10 &#181;L pipette tip. 
	NOTE: The scraping is completed when the surface of the membrane becomes uneven. The cell numbers from each membrane is variable (100 - 500 cells/membrane). Process the samples within 2 - 3 h of collection.</li>
</ol>
2. Immunophenotyping Cells by Flow Cytometry
<ol>
	<li>Centrifuge the cells at 400 x g for 5 min at room temperature to remove the media. Resuspend the cell pellet with 50 &#181;L of flow cytometry staining buffer (phosphate buffered saline (PBS) + 0.05% bovine serum albumin (BSA)).</li>
	<li>Stain cells with a panel of antibodies (e.g., CD3 brilliant violet (BV) 510 (UCHT1), CD4 allophycocyanine-H7 (APC-H7) (SK3), CCR7 phycoerythrin (PE)-A, CD45RO PE-cyanin 7 (PE-Cy7)-A, and live/dead cell mark 7-ADD) at room temperature for 20 - 30 min in the dark. Use antibodies directly from the stock according to the manufacturer&#39;s protocol. 
	NOTE: For the antibody concentration, use 2.5 &#181;L of CD3-BV510, 1.25 &#181;L of CD4-APC-H7, CD45RO-PE-Cy7, 7-AAD and 10 &#181;L of CCR7-PE. Use peripheral blood mononuclear cells (PBMCs) to titrate the different concentrations of antibodies. For the titration, take the antibody concentration mentioned in the manufacturer&#39;s protocol and use half and one-fourth of the recommended concentration. Use the minimum concentration of antibodies to avoid spill-over to other fluorochrome channels. Clear signals were observed with the mentioned concentrations of antibodies. The compensation was performed by testing PBMCs initially according to the protocol mentioned in Williams et al.20 For testing, PBMCs were incubated with the same set of antibodies used here and compensated by comparing single and multiple antibody stains.</li>
	<li>After the incubation period, add 1 mL of staining buffer to the tube, mix well, and centrifuge at 450 x g for 5 min at room temperature. Resuspend cells in 200 &#181;L of staining buffer.</li>
	<li>Run samples on a flow cytometry machine.
	<ol>
		<li>Before acquiring the data, calibrate the flow cytometer channel voltages with cytometer setup and tracking (CS&#38;T) beads to normalize the data acquisition on different days as per manufacturer&#39;s instructions.
		<ol>
			<li>Open the flow cytometry data collection software, click &#34;Set up &#38; QC&#34; button, choose the correct CS&#38;T bead lot number, load the beads, and then click the &#34;Start&#34; button.</li>
		</ol>
		</li>
		<li>Resuspend cells by gently tapping the tube before loading samples to the machine. Open the data collection software and click &#34;Preview&#34;. When threshold rate is stable, click &#34;Acquire&#34;. Monitor the sample level and click &#34;Stop&#34; when samples run out. 
		NOTE: Acquire 10,000 events per sample.</li>
		<li>After acquiring 10,000 events, generate a SSC-A and FSC-A dot plot in the worksheet.
		<ol>
			<li>Click the &#34;Polygon Gate&#34; button and draw a gate (this is P1) over all events with a FSC-A value &#62; 5 x 104 to exclude debris. Click &#34;Create dot plot&#34; button to generate a new dot plot, right click the dot blot, choose &#34;Properties&#34; to open the &#34;Plot Editor&#34; window, and then choose &#34;P1&#34;. Click the FSC-A on x-axis of P1 dot plot, change it to CD3-BV510. Draw a &#39;&#39;Polygon Gate&#39;&#39; to choose the CD3+ population and name it &#39;&#39;P2&#39;&#39;.</li>
			<li>Click &#34;Create dot plot&#34; button to generate a new dot plot, right click the dot blot, choose &#34;Properties&#34; to open the &#34;Plot Editor&#34; window. Click the FSC-A on x-axis of P2 dot plot and change it to 7-AAD. Draw a &#39;&#39;Polygon Gate&#39;&#39; for 7-AAD- population and choose &#39;&#39;P3&#39;&#39;. P3 here is CD3+ live cells.</li>
			<li>Click &#34;Create dot plot&#34; button to generate a new dot plot, right click the dot blot, choose &#34;Properties&#34; to open the &#34;Plot Editor&#34; window. Click the CD3-BV510 on x-axis and CD4-APCH7 on y-axis of the P3 dot plot. Draw Rectangle Gates on the upper and lower sides of the quadrant and choose &#39;&#39;P4&#39;&#39; and &#39;&#39;P5&#39;&#39;, respectively. P4 is live CD3+CD4+ and P5 is live CD3+CD4- T cells.</li>
			<li>Click &#34;Create dot plot&#34; button to generate a new dot plot, right click the dot blot, choose &#34;Properties&#34; to open the &#34;Plot Editor&#34; window. Click the CD45RO PE-Cy7 on the x-axis and CCR7-PE on the y-axis of both the P4 and P5 plots. Draw &#39;&#39;Quad Gate&#39;&#39; on this dot plot. 
			NOTE: Upper left hand side, upper right hand side, lower right hand side and lower left hand side quadrants here are na&#239;ve, central memory (TCM), effector memory (TEM), terminally differentiated effector memory (TEMRA) T cells, respectively.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Thatcher, R. W., Darougar, S., Jones, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjunctival+impression+cytology.">Conjunctival impression cytology.</a> Arch Ophthalmol. 95 (4), 678-681 (1977).</li><li>Egbert, P. R., Lauber, S., Maurice, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+conjunctival+biopsy.">A simple conjunctival biopsy.</a> Am J Ophthalmol. 84 (6), 798-801 (1977).</li><li>Baudouin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathology+of+dry+eye.">The pathology of dry eye.</a> Surv Ophthalmol. 45, S211-S220 (2001).</li><li>Nelson, J. D., Wright, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjunctival+goblet+cell+densities+in+ocular+surface+disease.">Conjunctival goblet cell densities in ocular surface disease.</a> Arch Ophthalmol. 102 (7), 1049-1051 (1984).</li><li>Brignole, F., 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=Expression+of+Fas-Fas+ligand+antigens+and+apoptotic+marker+APO2.7+by+the+human+conjunctival+epithelium.+Positive+correlation+with+class+II+HLA+DR+expression+in+inflammatory+ocular+surface+disorders.">Expression of Fas-Fas ligand antigens and apoptotic marker APO2.7 by the human conjunctival epithelium. Positive correlation with class II HLA DR expression in inflammatory ocular surface disorders.</a> Exp Eye Res. 67 (6), 687-697 (1998).</li><li>Knop, E., Knop, 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+role+of+eye-associated+lymphoid+tissue+in+corneal+immune+protection.">The role of eye-associated lymphoid tissue in corneal immune protection.</a> J Anat. 206 (3), 271-285 (2005).</li><li>Lopez-Miguel, A., Gutierrez-Gutierrez, S., Garcia-Vazquez, C., Enriquez-de-Salamanca, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Collection+From+Human+Conjunctival+Epithelial+Cells+Obtained+With+a+New+Device+for+Impression+Cytology.">RNA Collection From Human Conjunctival Epithelial Cells Obtained With a New Device for Impression Cytology.</a> Cornea. 36 (1), 59-63 (2017).</li><li>Tomlins, P., Roy, P., Cunow, J., Rauz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+EyePRIM+Device+for+Conjunctival+impression+for+Flow+Cytometry.">Assessment of the EyePRIM Device for Conjunctival impression for Flow Cytometry.</a> Invest Opthalm Vis Sci. 54, 5430-5430 (2013).</li><li>Barros Jde, N., Almeida, S. R., Lowen, M. S., Cunha, M. C., Gomes, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impression+cytology+in+the+evaluation+of+ocular+surface+tumors:+review+article.">Impression cytology in the evaluation of ocular surface tumors: review article.</a> Arq Bras Oftalmol. 78 (2), 126-132 (2015).</li><li>Calonge, 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=Impression+cytology+of+the+ocular+surface:+a+review.">Impression cytology of the ocular surface: a review.</a> Exp Eye Res. 78 (3), 457-472 (2004).</li><li>Haller-Schober, E. 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=Evaluating+an+impression+cytology+grading+system+(IC+score)+in+patients+with+dry+eye+syndrome.">Evaluating an impression cytology grading system (IC score) in patients with dry eye syndrome.</a> Eye (Lond). 20 (8), 927-933 (2006).</li><li>Singh, R., Joseph, A., Umapathy, T., Tint, N. L., Dua, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impression+cytology+of+the+ocular+surface.">Impression cytology of the ocular surface.</a> Br J Ophthalmol. 89 (12), 1655-1659 (2005).</li><li>Williams, G. P., 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=Conjunctival+Neutrophils+Predict+Progressive+Scarring+in+Ocular+Mucous+Membrane+Pemphigoid.">Conjunctival Neutrophils Predict Progressive Scarring in Ocular Mucous Membrane Pemphigoid.</a> Invest Ophthalmol Vis Sci. 57 (13), 5457-5469 (2016).</li><li>Best, N., Drury, L., Wolffsohn, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+evaluation+of+the+Oculus+Keratograph.">Clinical evaluation of the Oculus Keratograph.</a> Cont Lens Anterior Eye. 35 (4), 171-174 (2012).</li><li>Downie, L. E., Keller, P. R., Vingrys, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+ocular+bulbar+redness:+a+comparison+of+methods.">Assessing ocular bulbar redness: a comparison of methods.</a> Ophthalmic Physiol Opt. 36 (2), 132-139 (2016).</li><li>Amparo, F., Wang, H., Emami-Naeini, P., Karimian, P., Dana, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ocular+Redness+Index:+a+novel+automated+method+for+measuring+ocular+injection.">The Ocular Redness Index: a novel automated method for measuring ocular injection.</a> Investigative ophthalmology &amp; visual science. 54 (7), 4821-4826 (2013).</li><li>Shapiro, A., Merin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schirmer+test+and+break-up+time+of+tear+film+in+normal+subjects.">Schirmer test and break-up time of tear film in normal subjects.</a> American journal of ophthalmology. 88 (4), 752-757 (1979).</li><li>Finis, D., Pischel, N., Konig, C., Hayajneh, J., Borrelli, M., Schrader, S., Geerling, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+OSDI+and+SPEED+questionnaires+for+the+evaluation+of+dry+eye+disease+in+clinical+routine.">Comparison of the OSDI and SPEED questionnaires for the evaluation of dry eye disease in clinical routine.</a> Ophthalmologe. 111 (11), 1050-1056 (2014).</li><li>Behrens, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfunctional+tear+syndrome:+a+Delphi+approach+to+treatment+recommendations.">Dysfunctional tear syndrome: a Delphi approach to treatment recommendations.</a> Cornea. 25 (8), 900-907 (2006).</li><li>Williams, G. P., Pachino, A., Long, H. M., Rauz, S., Curnow, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+production+and+antigen+recognition+by+human+mucosal+homing+conjunctival+effector+memory+CD8++T+cells.">Cytokine production and antigen recognition by human mucosal homing conjunctival effector memory CD8+ T cells.</a> Invest Opthalmol Vis Sci. 55 (12), 8523-8530 (2014).</li><li>Bose, T., Lee, R., Hou, A., Louis, T., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+resident+memory+T+cells+in+the+human+conjunctiva+and+immune+signatures+in+human+dry+eye+disease.">Tissue resident memory T cells in the human conjunctiva and immune signatures in human dry eye disease.</a> Sci Rep. 7, 45312 (2017).</li><li>Williams, G. P., 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=The+dominant+human+conjunctival+epithelial+CD8alphabeta++T+cell+population+is+maintained+with+age+but+the+number+of+CD4++T+cells+increases.">The dominant human conjunctival epithelial CD8alphabeta+ T cell population is maintained with age but the number of CD4+ T cells increases.</a> Age (Dordr). 34 (6), 1517-1528 (2012).</li><li>Mrugacz, M., Zak, J., Bakunowicz-Lazarczyk, A., Wysocka, J., Minarowska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+HLA-DR+antigen+in+conjunctival+epithelial+cells+of+patients+with+cystic+fibrosis.">Flow cytometric analysis of HLA-DR antigen in conjunctival epithelial cells of patients with cystic fibrosis.</a> Eye (Lond). 21 (8), 1062-1066 (2007).</li></ol>

The authors declare no competing financial interests.

This is an easy, quick and less invasive technique that can be used in the out-patient clinics for relatively fast immune profiling in contrast to the conventional techniques like scraping, swabbing, pipetting or absorbent filter paper1,2. A variant of this technique is already being used in research settings22. The future application of the proposed methodology is for patient stratification in clinical trials with ocular diseases, especially those requiring immunophenotyping.
A major challenge with this technique is the relatively few immune cells retrieved after impression collection and scraping. The total number of CD3+ T cells recovered from four impressions per individual varied from ~ 500 - 1,000 cells. The ocular samples were washed before the flow cytometry analysis for a minimal number of times to avoid further loss of cells. Critical steps and challenges that remain within the protocol are efficient collection of ocular samples and proper scraping of the membrane to achieve higher cell numbers. Nevertheless, this limitation is unlikely to bias towards any specific immune phenotype. The troubleshooting performed here to maximize the yield of cells was to reduce the number of washing steps after and before the incubation of antibodies.
There are other limitations to using IC. In patients with severely keratinized or fibrosed ocular surface such as in Steven Johnson syndrome, the cellular yield may be even less than that in this study. The proportion of immune cells may change if the samples are stored instead of analyzed on the same day. It is difficult to predict if certain cell types are more resistant to storage than others. Previous studies have reported elevated levels of HLA-DR expression in the conjunctival epithelial cells23, so it would be interesting to evaluate the correlation between HLA-DR and levels of specific immune cells. Immune cells may also be associated with the expression level of chemokines. These issues should be addressed in future studies.

Impression cytology (IC) was first performed in 1977 by Thatcher et al1. They used a plastic impression disc to collect conjunctival cells from patients instead of other techniques available at that time such as scraping, swabbing or pipetting1. The current technique of IC uses an absorbent filter paper2 to imprint the bulbar and palpebral conjunctiva and collect the most superficial layer of conjunctival cells. These cells, having reached their final stage of differentiation, are continuously shed into tears3. Three major populations of cells are found in IC specimens: epithelial cells4, goblet cells3,5, and mucosal-associated lymphoid tissue viz epithelium-associated effector T cells or dendritic cells6. Ocular surface cells in IC samples can be analyzed by microscopy, immune-blotting and reverse-transcriptase polymerase chain reaction (RT-PCR)7. Flow cytometry has been recently used to analyze immune cells collected by scraping the IC membrane8. Interestingly, IC6,9 has been used to evaluate many ocular surface diseases including keratoconjunctivitis sicca, vitamin A deficiency, cicatricial pemphigoid, atopic disease, superior limbic keratoconjunctivitis, vernal keratoconjunctivitis, and epithelial squamous metaplasia. IC has also been used to evaluate the impact of wearing contact lenses, detecting ocular surface microbes, and testing therapeutic efficacy and tolerance of therapeutic interventions in longitudinal studies10,11,12.
The medical device (EyePrim) is supported by a type of polyethersulfone (PES) 0.2-&#181;m membrane, which has been previously validated for the technique of ocular impression cytology with flow cytometry (OSIC-flow) and opens up opportunities to use longitudinal sampling to monitor disease progression and response to treatment (e.g., the detailed analysis of intraepithelial leukocytes defined as putative disease markers for progressive conjunctival fibrosis in mucous membrane pemphigoid)13. Early researchers used autoclaved PES filters that required manual impression. As a result, the yield was variable and user dependent. The advantage of this medical device is ease of use, standardized pressure (Pa or N/m2), and enables repeatability, reproducibility, and consistent cellular recovery. This technique is useful in an out-patient clinic because it is non-surgical, easy-to-perform and rapid. This is a Class I (sterile) medical device according to the directive 93/42/CEE, CE 0499 (SNCH). It only requires topical anesthesia during the procedure, which ensures maintenance of the integrity of the ocular surface. Following IC, cells can be processed immediately for flow cytometry. Moreover, it is possible for non-ophthalmology technicians and nurses to be trained to sample the ocular surface.
Despite the improvement of IC over other techniques, several challenges remain. For example, there may be variation due to the area of the sampling and regional differences in the bulbar conjunctiva depending on the position of the IC. Another source of variation is due to the application of different amounts of pressure during IC. Other methodological issues involve standardization of cell processing: these involve duration and method of fixation, and the conditions of possible storage, which may impact stability of the sampled material.
The overall goal of this technique is to develop a method of isolation of the ocular impression samples that is easy to use, non-invasive and can be applied to the immunological characterization of the clinical samples.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-human CD3 BV510</td><td>BD Biosciences</td><td>563109</td><td></td></tr><tr><td>anti-human CD4 APCH7</td><td>BD Biosciences</td><td>641398</td><td></td></tr><tr><td>anti-human CD45RO PECy7</td><td>BD Biosciences</td><td>337168</td><td></td></tr><tr><td>7-AAD solution</td><td>BD Biosciences</td><td>555816</td><td></td></tr><tr><td>anti-human CCR7 PE</td><td>BD Biosciences</td><td>552176</td><td></td></tr><tr><td>Pippetes</td><td>Eppendorf</td><td>NA</td><td></td></tr><tr><td>Local Anaesthesia</td><td>Alcaine</td><td>NA</td><td></td></tr><tr><td>Fluorescein sodium solution</td><td>Bausch &amp;amp; Lomb U.K Limited</td><td>NA</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Keratograph 5M</td><td>Oculus</td><td>NA</td><td></td></tr><tr><td>Slit lamp BioMicroscope</td><td>Haag Streit</td><td>BM900</td><td></td></tr><tr><td>EyePrim</td><td>Opia Technologies</td><td>NA</td><td></td></tr><tr><td>FACS Verse</td><td>BD BioSciences</td><td>NA</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Softwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GraphPad 6.0</td><td>Prism</td><td>NA</td><td></td></tr><tr><td>FACSVerse analysis software</td><td>BD</td><td>NA</td><td></td></tr></tbody></table>

a non-invasive way to isolate and phenotype cells from the conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

IC by this clinical device allowed us to isolate ocular surface immune cells while keeping the ocular surface intact. Figure 1 describes how the IC was performed. The different sites of the eye from where the samples were collected are marked. Figure 2 shows representative result of flow cytometry collected from 10 healthy controls. For the gating, use SSC and 7-AAD to distinguish between live and dead cells. 7-AAD- cells are identified as live cells. These live cells are further characterized by CD3+ and CD4+ markers. Furthermore, the CD4+ and CD4- populations were each further characterized as Na&#239;ve, TEM, TCM and TEMRA with the CCR7 and CD45RO markers. Amongst CD3+ T cells, effector memory T cells predominate in the human ocular surface. In earlier experiments, it has also been shown that CD8+ memory cells are the major populations in the conjunctival epithelial T cells among the healthy individuals20. Figure 3 shows that CD4+ and CD8+ effector memory T cells (TEM and TEMRA) are the major subset in the human ocular surface in 10 healthy controls studied. Each population mentioned in the figure are notified with their marker phenotype and names (Na&#239;ve, TCM, TEM, TEMRA). The numbers in red denote the percentage of the population. The data in this figure are represented as mean &#177; SEM
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55591/55591fig1.jpg" /> 
Figure 1: Collection of IC Samples from the Ocular Surface by the Impression Cytology Device. Samples were collected from the temporal bulbar region as shown.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55591/55591fig2.jpg" /> 
Figure 2: Dot Plot Graph using Flow Cytometry. Live 7-AAD- cells were selected. The CD3+ cells were first gated, and then this population was gated into CD4+ and CD4- subsets. The proportions of na&#239;ve (CCR7+CD45RO-), central memory (CCR7+CD45RO+) and effector memory (CCR7-CD45RO+; CCR7-CD45RO-) subsets was determined with the help of CCR7 and CD45RO markers. <a href="http://cloudflare.jove.com/files/ftp_upload/55591/55591fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55591/55591fig3.jpg" /> 
Figure 3: Immune Cell Sub-types in CD4+ and CD8+ T Cells of Healthy Human Ocular Surface. Distribution of conjunctival CD4+ and CD8+ na&#239;ve, central memory (TCM), and effector memory (TEM and TEMRA) subsets in healthy human controls; each data point represents a separate individual mean &#177; SEM shown. The percentage of the population is marked as red below the name of each of the populations. The total percentage of the population is 98%, because CD4+ TEMRA population was not included in the mentioned scatterplot (modified from Bose et al.21). <a href="http://cloudflare.jove.com/files/ftp_upload/55591/55591fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva at JoVE.com

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

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Research

JoVE Journal

Immunology and Infection

10.3K Views.  Nanyang Technological University.  Het blootgestelde normale oculaire oppervlak bestaat uit hoornvlies en conjunctiva. Epitheliale cellen, spekcellen en immuuncellen zijn aanwezig in de conjunctiva. Hier wordt een niet-invasieve techniek van impressie cytologie beschreven met behulp van een impressie cytologie apparaat en flow cytometrie om immuuncellen in de conjunctiva te analyseren. 

Video: A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Bioengineering

Impression Cytology of the Lid Wiper Area

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.

We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort.

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The ...

Medicine

Conjunctival Commensal Isolation and Identification in Mice

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an &#8220;in-lab&#8221; made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular ...

A Protocol to Isolate, Culture, and Identify Human Limbal Niche Cells

Isolation and Identification of Limbal Niche Cells

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 &#176;C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal&#160;epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 &#176;C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFR&#946;). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFR&#946;, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were&#160;68%, 99%, 20%, and 3% in&#160;BMMSCs. The standard process for LNC&#160;isolation and identification could provide&#160;a reliable laboratory basis for the widespread use of LNCs.

Author Spotlight: Standardizing Limbal Niche Cell (LNC) Isolation and Characterization to Support Widespread LNC Research 

Here, we present a protocol to isolate and identify the human limbal niche cells.

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used ...

Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. Disruption of OS would lead to keratitis, conjunctivitis or both (keratoconjunctivitis). In experimental animal models with certain genetic modifications or artificial operations, it is useful to examine all parts of the OS epithelial sheet to evaluate relative pathogenetic changes of each part in parallel. However, dissection of OS tissue as a whole without distortion or damage has been challenging, primarily due to the softness and thinness of the OS affixed to physically separate yet movable eyelids and eyeball. Additionally, the deep eye socket formed by the hard skull/orbital bones is fully occupied by the eyeball leaving limited space for operating dissections. As a result, direct dissection of the eyeball with associated OS tissues from the facial side would often lead to tissue damages, especially the palpebral and bulbar conjunctiva. In this protocol, we described a method to remove the skull and orbital bones sequentially from a bisected mouse head, leaving eyelids, ocular surface, lens and retina altogether in one piece. The integrity of the OS sheet was well preserved and could be examined by histology or immunostaining in a single section.

This protocol describes a method for the isolation of the mouse eyeball with eyelid, ocular surface, anterior and posterior segments in relatively intact position.

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. ...

Biology

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.

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.

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

Isolating IPE and RPE Cells: A Technique for Obtaining Ocular Pigment Epithelial Cells from Rabbit Eye

Source: Bascuas, T., et al. <a href="https://www.jove.com/v/62145">Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy.</a> J. Vis. Exp. (2021).
In this video, we show a procedure to obtain ocular pigment epithelial cells from a rabbit model. The cells are helpful in developing therapeutic strategies for retinal degenerative diseases.

Source: Bascuas, T., et al. Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy. J. Vis. ...

JoVE Encyclopedia of Experiments

Large Animal Models

Leporine

Swab-based Conjunctival Commensal Bacteria Isolation: A Technique to Isolate Commensal Bacteria from Conjunctiva of Murine Model

Source: Smith-Page, K. et al. <a href="https://www.jove.com/video/61672" target="_blank">Conjunctival Commensal Isolation and Identification in Mice</a>. J. Vis. Exp. (2021)
This video describes a swab-based technique to isolate viable commensal bacteria from the ocular conjunctiva. The technique can be used to profile the commensal bacterial microbiome of the conjunctiva, which plays a defensive role against pathogens.

Source: Smith-Page, K. et al. Conjunctival Commensal Isolation and Identification in Mice. J. Vis. Exp. (2021)
This video describes a swab-based technique ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

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