Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R01CA174385. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Reagents Preparation
<ol>
 <li>Prepare RPMI-PLGH by mixing 500 ml RPMI-1640 and 5 ml each of Penicillin-streptomycin, L-glutamine, and HEPES solution.</li>
 <li>Prepare R10 solution by mixing RPMI-PLGH with 10% Fetal Bovine Serum (FBS). The FBS is heat-inactivated at 56 &deg;C for 30 min prior to addition.</li>
 <li>Pre-warm at 37 &deg;C 50 ml of RPMI-PLGH, 15 ml of PBS, and 15 ml of R10 in sterile conical tubes.</li>
 <li>Fabrication of arrays of nanowells in PDMS: The silicon master is fabricated using photolithography essentially as described previously10. Mix thoroughly the Sylgard 184 elastomer kit base and curing agent at 10:1 weight ratio in a disposable cup using a plastic knife. Degas the mixture in a vacuum chamber for 1 hr. Pour mixture onto the nanowell array, seal with a glass slide and let it sit for 30 min. Transfer the assembly into an oven set to 80 &deg;C for 2 hr and let cool at room temperature for 1 hr. The elastomer will cure during this period and will bond to the glass slide. The glass slide containing the PDMS nanowell array is carefully lifted off the silicon master, covered with Scotch tape and stored until future use.</li>
</ol>
2. Target Cell (T) Labeling
<ol>
 <li>Count 5 million target cells from cell culture using a hemocytometer and pellet the cells in a sterile 15 ml conical tube by centrifugation at 340 x g for 5 min. The cell culture is split the previous day in 5 ml total volume (density = 1 million/ml) and cultured in T-25 flask (VWR). Protocols for cell counting using hemocytometer have been described in detail previously11</li>
 <li>Aspirate excess media leaving behind approximately 50 &mu;l of media.</li>
 <li>Prepare a working solution of Cell Tracker Red (CTR) by mixing 0.5 &mu;l CTR stock (1 mM) to 150 &mu;l RPMI-PLGH (final concentration 2.5 &mu;M). Add 150 &mu;l of the working solution to the target cells and mix thoroughly using a P200 pipette. Alternately, PKH 26 (final concentration of 2 &mu;M) can be used label target cells as per the manufacturer instructions.</li>
 <li>Incubate at 37 &deg;C/5% CO2 for 20 min.</li>
</ol>
3. Effector (E) Cell Labeling
<ol>
 <li>Count 5 million effector cells from cell culture using a hemacytometer and pellet the cells in a sterile 15 ml conical tube by centrifugation at 340 x g for 5 min.</li>
 <li>Aspirate excess media</li>
 <li>Prepare a working solution of Vybrant DyeCycleViolet Stain solution by adding 1 &mu;l of 5 mM Vybrant Violet stock to 1 ml of R10 media (final concentration of 5 &mu;M). Add the working solution to the cells and resuspend using P200 pipette. Alternately, PKH 67 (final concentration of 2 &mu;M) can be used label target cells as per the manufacturer instructions. If PKH 67 is used, then SYTOX Green should not be used since they are in the same fluorescence channel. The enumeration of apoptotic targets is done exclusively using AnnexinV staining in this case. 
 Note: If performing time-lapse and not endpoint measurements, UV dyes like Vybrant Violet should be avoided.</li>
 <li>Incubate at 37 &deg;C/5% CO2 for 20 min.</li>
</ol>
4. Separation of Live and Dead Cells Using a Density Gradient
<ol>
 <li>Add 6.8 ml and 6.0 ml of RPMI-PLGH to target and effector cells, respectively and mix by pipetting up and down gently.</li>
 <li>Layer 3.5 ml of FICOLL-Paque Plus to bottom of each conical tube containing target and effector cells. Layer the FICOLL slowly to ensure good formation of layers between FICOLL and media.</li>
 <li>Centrifuge both tubes at 340 x g for 30 min. with no brake and acceleration.</li>
 <li>During centrifugation, transfer 7 ml of pre-warmed PBS to a 4-well plate. Prepare nanowell array (fabricated in PDMS): clean bottom of glass slide with ethanol and oxidize the PDMS at high RF setting using plasma oxidizer for 1 min. This step will sterilize the nanowell array while also rendering the PDMS hydrophilic. Immediately move the nanowell array into the plate containing PBS.</li>
 <li>Transfer the plate to a biosafety cabinet and aspirate excess PBS. Make 10 ml 1% noble agar by adding 100 mg agar powder to 10 ml DI water and then microwave it for 30-45 sec. Apply warm 1% noble agar on top and bottom edge of glass slide that contains the nanowell array to immobilize the array and wait 10 min for agar to solidify. Add 3 ml PBS and aspirate excess agar.</li>
 <li>Add 3 ml of R10 onto the top of nanowell array and let it equilibrate for ~10 min.</li>
 <li>Subsequent to FICOLL centrifugation, aspirate 5 ml media from the top layer of each tube. Be careful not to aspirate the layer containing the cells.</li>
 <li>Harvest the cells (white layer) by recovering 1-2 ml from the layer between FICOLL and media with P1,000 pipette and move to new sterile conical tubes. Repeat this for both sets of cells while making sure that you maximize the recovery cells.</li>
 <li>Add 3 ml of pre-warmed RPMI-PLGH to each tube, mix by pipetting and pellet by centrifugation at 340 x g for 5 min on full acceleration and brake.</li>
 <li>Aspirate excess media, add 5 ml of pre-warmed RPMI-PLGH to each tube and repeat centrifugation at 340 x g for 5 min.</li>
 <li>Aspirate excess media and resuspend the cell pellet in 500 &mu;l of R10.</li>
 <li>Count live cells using the Trypan blue exclusion method on a hemacytometer.</li>
</ol>
5. Cell Loading onto Nanowell Array
<ol>
 <li>Aspirate excess media from nanowell array and add 2 ml of fresh R10 across the array. Aspirate again while making sure the top of nanowell array is not too wet/dry as it will affect distribution of cells.</li>
 <li>Deposit 1 x 105 effector cells in 200 &mu;l of R10 onto nanowell array surface (density = 5 x 105 cells/ml).</li>
 <li>Let cells settle for 5 min and using a standard tissue culture microscope check to ensure that the distribution of cells is desirable. Add more cells if necessary.</li>
 <li>Deposit 1 x 105 target cells in 200 &mu;l R10 on nanowell array surface (density = 5 x 105 cells/ml).</li>
 <li>Let cells settle for 5 min and using a standard tissue culture microscope check to ensure that the distribution of cells is desirable. Add more cells if necessary.</li>
 <li>Rinse nanowell array carefully with 2 ml R10 and aspirate excess media.</li>
 <li>Prepare 3 ml pre-warmed R10 in a 15 ml sterile conical tube. Add 3 &mu;l of 0.5mM SYTOX Green Nucleic Acid Stain (final concentration 0.5 &mu;M) and 60 &mu;l of Annexin V- Alexa Fluor 647 to prepare working solution. Add the working solution onto the 4-well plate carefully while making sure that cells are not displaced from the wells.</li>
 <li>Incubate at 37 &deg;C/5% CO2 for 15 min.</li>
</ol>
6. Imaging 1
<ol>
 <li>Acquire images of the nanowell array using a fluorescence microscope: In this example, we use a ZEISS Observer Z-1 microscope equipped with a motorized stage and Lambda-DG4.</li>
 <li>Initialize the software and the microscope and check the signal intensity on the transmitted light and the other fluorescent channels. Overall, there will be five channels corresponding to: transmitted light, Annexin V-647 (Cy5 filter), CTR (DsRed filter), SYTOX Green (FITC filter), and Vybrant Violet (DAPI filter). Ensure that the microscope it setup to ensure maximal signal to noise while avoiding saturation.</li>
 <li>Set X and Y position for the nanowell array. Begin process by setting the zero position and initializing focus on the center of 7 x 7 nanowell array block on top left corner of the nanowell array stamp. Set the x,y positions to coincide with the center of each block and the z-value to accurately reflect the focus on each block.</li>
 <li>Once the position and focus are all set, make sure to set image acquisition in linear mode and acquire images. Note: If necessary turn on the incubator and CO2 on the microscope at least 30 min before the experiment.</li>
</ol>
7. Post-imaging
Transfer the 4-well plate containing the nanowell array into an incubator (37 &deg;C/5% CO2) for 6 hr.
8. Imaging 2
Acquire the images at the second time-point as outlined in Step 6.
9. Image Analysis
<ol>
 <li>The occupancy of cells within the nanowells is determined by the use of appropriate image segmentation programs (e.g. ImageJ, <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>) essentially as described previously9,12 .</li>
 <li>Analysis of the initial set of microscope images (t = 0 hr; t0) is used to generate a database of the individual nanowells containing exactly one live target cell (identified by CellTracker Red; red) and no dead cells (identified by Annexin V staining, green) [T1t0 nanowells].</li>
 <li>Analysis of the second set of microscope images (t =6 hr; t6) is used to independently determine a second database of nanowells containing 1 target cell (red) [T1t6 nanowells]. An integrity check (database query) is implemented to ensure that all live targets at t0 are matched to targets at t6 to produce a final set of matched targets (implemented as nanowells that have T1t0 = T1t6, designated as T1match)</li>
 <li>The number of effector cells in nanowells is determined using Vybrant Violet staining at t0 [E1 nanowells]</li>
 <li>A database query is implemented to identify nanowells containing single effectors co-incubated with single targets (defined as nanowells that have E1 and T1match, designated as E1T1)</li>
 <li>Effector induced apoptosis is scored by identifying targets that are Annexin V negative at t0 and Annexin V positive at t4 (E1T1 with Annexin V target staining at t6).</li>
</ol>
10. Optional Time-lapse Imaging of Killing Using Nikon's BioStation IM
<ol>
 <li>Take a Petri dish with coverslip bottom and cut out a small piece of the nanowell array chip (prepared at Step 5) that will exactly fit on the coverslip. Make sure that the chip stays wet while cutting.</li>
 <li>Mix 1 x 106 targets and 1 x 106 T cells in 100 &mu;l media, and pipette it onto the coverslip.</li>
 <li>Allow about 15-30 min to let the cells settle.</li>
 <li>Rapidly invert the small nanowell array chip and place it on the coverslip.</li>
 <li>In order to ensure that the chip will not be able to move during the imaging, place one or two 20mm x 20mm coverslips onto the top of the chip. Apply pressure softly to push the chip to the bottom of the dish.</li>
</ol>
Note: The nanowell array chip's bottom is too thick for high-resolution imaging and therefore has to be turned upside down. During this, many of the cells are washed out. Since this cannot be easily controlled, cell numbers in Step 9.2 and incubation time in Step 9.3 have to be optimized.

<ol>
	<li>Louis, C. U., 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=Antitumor+activity+and+long-term+fate+of+chimeric+antigen+receptor-positive+T+cells+in+patients+with+neuroblastoma.">Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma.</a> Blood. 118, 6050-6056 (2011).</li><li>Savoldo, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD28+costimulation+improves+expansion+and+persistence+of+chimeric+antigen+receptor-modified+T+cells+in+lymphoma+patients.">CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients.</a> J. Clin. Invest. 121, 1822-1826 (2011).</li><li>Kalos, 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=T+cells+with+chimeric+antigen+receptors+have+potent+antitumor+effects+and+can+establish+memory+in+patients+with+advanced+leukemia.">T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.</a> Sci. Transl. Med. 3, 95ra73 (2011).</li><li>Porter, D. L., Levine, B. L., Kalos, M., Bagg, A., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+Antigen+Receptor-Modified+T+Cells+in+Chronic+Lymphoid+Leukemia.">Chimeric Antigen Receptor-Modified T Cells in Chronic Lymphoid Leukemia.</a> N. Engl. J. Med. , (2011).</li><li>Restifo, N. P., Dudley, M. E., Rosenberg, S. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy:+visualizing+immunity+in+context.">Intravital microscopy: visualizing immunity in context.</a> Immunity. 21, 315-329 (2004).</li><li>Lamoreaux, L., Roederer, M., Koup, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+cytokine+optimization+and+standard+operating+procedure.">Intracellular cytokine optimization and standard operating procedure.</a> Nat. Protoc. 1, 1507-1516 (2006).</li><li>Varadarajan, N., 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=A+high-throughput+single-cell+analysis+of+human+CD8++T+cell+functions+reveals+discordance+for+cytokine+secretion+and+cytolysis.">A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis.</a> J. Clin. Invest. 121, 4322-4331 (2011).</li><li>Ogunniyi, A. O., Story, C. M., Papa, E., Guillen, E., Love, 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=Screening+individual+hybridomas+by+microengraving+to+discover+monoclonal+antibodies.">Screening individual hybridomas by microengraving to discover monoclonal antibodies.</a> Nat. Protoc. 4, 767-782 (2009).</li><li>Streeck, H., Frahm, N., Walker, B. D. <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+IFN-gamma+Elispot+assay+in+HIV+vaccine+research.">The role of IFN-gamma Elispot assay in HIV vaccine research.</a> Nat. Protoc. 4, 461-469 (2009).</li><li>Varadarajan, N., 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=Rapid,+efficient+functional+characterization+and+recovery+of+HIV-specific+human+CD8++T+cells+using+microengraving.">Rapid, efficient functional characterization and recovery of HIV-specific human CD8+ T cells using microengraving.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 3885-3890 (2012).</li><li>Makedonas, G., Betts, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+in+a+house+of+cards:+re-evaluating+CD8++T-cell+immune+correlates+against+HIV.">Living in a house of cards: re-evaluating CD8+ T-cell immune correlates against HIV.</a> Immunol. Rev. 239, 109-124 (2011).</li><li>Telford, W. G., Komoriya, A., Packard, B. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+analysis+of+apoptosis+by+flow+and+image+cytometry.">Multiparametric analysis of apoptosis by flow and image cytometry.</a> Methods Mol. Biol. 263, 141-160 (2004).</li></ol>

No conflicts of interest declared.

We have outlined the protocol for a high-throughput single-cell cytolytic assay enabled via co-incubation of effectors and targets in arrays of nanowells (Figure 1). In addition to throughput a major advantage of the technique is the ability to monitor effector-mediated cytotoxicity against desired target cells without the need for target cell engineering which in turn allows for the use of autologous or matched/primary tumor cells as target cells. The spatial confinement allows the retrieval and ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>RPMI-1640 w/o L-glutamine</td>
 <td>Cellgro</td>
 <td>15-040-CV</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>Cellgro</td>
 <td>30-002-CI</td>
 <td>10,000 I.U. Penicillin 10,000 &mu;g/ml Streptomycin</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Cellgro</td>
 <td>25-005-CI</td>
 <td>200 mM solution</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma Aldrich</td>
 <td>H3537</td>
 <td>1M</td>
 </tr>
 <tr>
 <td>Fetal bovine serum (FBS)</td>
 <td>Atlanta Biologicals</td>
 <td>S11150</td>
 <td>Lot tested</td>
 </tr>
 <tr>
 <td>Cell Tracker Red Stain</td>
 <td>Invitrogen</td>
 <td>C34552</td>
 <td>50 &mu;g</td>
 </tr>
 <tr>
 <td>Vybrant DyeCycle Violet Stain</td>
 <td>Invitrogen</td>
 <td>V35003</td>
 <td>5 mM</td>
 </tr>
 <tr>
 <td>SYTOX green Nucleic Acid Stain</td>
 <td>Invitrogen</td>
 <td>S7020</td>
 <td>5 mM</td>
 </tr>
 <tr>
 <td>Annexin V-Alexa Fluor 647</td>
 <td>Invitrogen</td>
 <td>A23204</td>
 <td>500 &mu;l</td>
 </tr>
 <tr>
 <td>Dulbecco's PBS</td>
 <td>Cellgro</td>
 <td>21-031-CV</td>
 <td>500 ml</td>
 </tr>
 <tr>
 <td>Noble agar</td>
 <td>DIFCO</td>
 <td>2M220</td>
 <td>100 g</td>
 </tr>
 <tr>
 <td>Trypan Blue</td>
 <td>Sigma Aldrich</td>
 <td>T8154</td>
 <td>0.4% liquid, sterile filtered</td>
 </tr>
 <tr>
 <td>Hemocytometer</td>
 <td>Hausser Scientifics</td>
 <td>1492</td>
 <td>Bright line</td>
 </tr>
 <tr>
 <td>4-well plate</td>
 <td>Thermo Fisher</td>
 <td>167603</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Harrick Plasma Cleaner</td>
 <td>Harrick Plasma</td>
 <td>PDC-32G</td>
 <td>Basic plasma cleaner</td>
 </tr>
 <tr>
 <td>Observer.Z1</td>
 <td>ZEISS</td>
 <td>&nbsp;</td>
 <td>Fluorescent microscope (works with the three parts below)</td>
 </tr>
 <tr>
 <td>Lambda 10-3</td>
 <td>Sutter Instrument</td>
 <td>&nbsp;</td>
 <td>Filter controller</td>
 </tr>
 <tr>
 <td>Lambda DG-4</td>
 <td>Sutter Instrument</td>
 <td>&nbsp;</td>
 <td>Ultra-High-Speed Wavelength switcher</td>
 </tr>
 <tr>
 <td>Hamamatsu EM-CCD Camera</td>
 <td>Hamamatsu</td>
 <td>C9100-13</td>
 <td>CCD-Microscope camera</td>
 </tr>
 <tr>
 <td>15 ml conical tube</td>
 <td>BD Falcon</td>
 <td>352097</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 ml conical tube</td>
 <td>VWR</td>
 <td>3282-345-300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nikon Biostation</td>
 <td>Nikon Instruments Inc.</td>
 <td>Biostation IM</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass bottom culture dish</td>
 <td>MatTek Corporation</td>
 <td>P35G-0</td>
 <td>35 mm petri dish, 10 mm microwell</td>
 </tr>
 </tbody>
</table>

quantitative high-throughput single-cell cytotoxicity assay for t cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

An example of the application of the high-throughput cytolytic assay is demonstrated in Figure 2. Briefly, labeled CD19-specific CAR+ T cells were co-incubated with labeled mouse EL4 target cells in the individual wells of a nanowell array (Sections 1-5). An initial image was recorded on the automated fluorescent microscope to identify the occupancy (effectors and/or targets) of every single nanowell on the array (Section 6). Image processing was used to identify all nanowells containing exact...

Watch this Scientific Journal Video about Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells at JoVE.com

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

quantitative-high-throughput-single-cell-cytotoxicity-assay-for-t

Research

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Immunology and Infection

14.9K Views.  University of Houston. We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Video: Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...