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 border="0" cellpadding="0" cellspacing="0" width="940">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Reagents</td>
			<td style="width:300px;"></td>
			<td style="width:143px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">anti-human CD3 BV510</td>
			<td style="width:300px;">BD Biosciences</td>
			<td align="right">563109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">anti-human CD4 APCH7</td>
			<td style="width:300px;">BD Biosciences</td>
			<td align="right">641398</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">anti-human CD45RO PECy7</td>
			<td style="width:300px;">BD Biosciences</td>
			<td align="right">337168</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">7-AAD solution</td>
			<td style="width:300px;">BD Biosciences</td>
			<td align="right">555816</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">anti-human CCR7 PE</td>
			<td style="width:300px;">BD Biosciences</td>
			<td align="right">552176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Pippetes</td>
			<td style="width:300px;">Eppendorf</td>
			<td style="width:143px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Local Anaesthesia</td>
			<td style="width:300px;">Alcaine</td>
			<td style="width:143px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Fluorescein sodium solution</td>
			<td style="width:300px;">Bausch &#38; Lomb U.K Limited</td>
			<td style="width:143px;">NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:331px;">Name</td>
			<td style="width:300px;">Company</td>
			<td style="width:143px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Equipments</td>
			<td style="width:300px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Keratograph 5M</td>
			<td>Oculus</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Slit lamp BioMicroscope</td>
			<td>Haag Streit</td>
			<td>BM900</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">EyePrim</td>
			<td>Opia Technologies</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">FACS Verse</td>
			<td style="width:300px;">BD BioSciences</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:331px;">Name</td>
			<td style="width:300px;">Company</td>
			<td style="width:143px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Softwares</td>
			<td style="width:300px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">GraphPad 6.0</td>
			<td style="width:300px;">Prism</td>
			<td style="width:143px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">FACSVerse analysis software</td>
			<td style="width:300px;">BD</td>
			<td style="width:143px;">NA</td>
			<td></td>
		</tr>
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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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.2K Views.  Nanyang Technological University. 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.

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...