We are grateful for the scientific discussions with Dr. Benjamin Chen. This work was funded by K08AI120806 (THS), R01DA052255 (THS and KB), and R21AI152833 (THS and KB).

1. Preparation of cells for imaging
<ol>
	<li>Prepare fresh Fluo-4 AM loading solution: Add 25 &#181;L of 100x concentrated detergent solvent, then add 2.5 &#181;L of Fluo-4 AM 1000x to a 1.5 mL tube. Vortex to mix.</li>
	<li>Pipette 2.5 mL of culture media into the loading solution and invert to mix. Protect from light.</li>
	<li>Centrifuge 2 x 106 MT-4 cells at 500 x g for 3 min.</li>
	<li>Remove media and resuspend the pellet in 2 mL of the prepared Fluo-4 AM loading solution. Protect from light....

<ol>
	<li>Aberg, 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=Aging,+inflammation,+and+HIV+infection.">Aging, inflammation, and HIV infection.</a> Topics in Antiviral Medicine. 20 (3), 101-105 (2012).</li><li>Deeks, S. G., Tracy, R., Douek, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+effects+of+inflammation+on+health+during+chronic+HIV+infection.">Systemic effects of inflammation on health during chronic HIV infection.</a> Immunity. 39 (4), 633-645 (2013).</li><li>Swartz, T. H., Dubyak, G. R., Chen, B. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+receptors+are+required+for+HIV-1+infection+of+primary+human+macrophages.">Purinergic receptors are required for HIV-1 infection of primary human macrophages.</a> Journal of Immunology. 188 (9), 4488-4495 (2012).</li><li>Marin, 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=High-throughput+HIV-cell+fusion+assay+for+discovery+of+virus+entry+inhibitors.">High-throughput HIV-cell fusion assay for discovery of virus entry inhibitors.</a> Assay and Drug Development Technology. 13 (3), 155-166 (2015).</li><li>Soare, A. Y., 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=P2X+Antagonists+inhibit+HIV-1+productive+infection+and+inflammatory+cytokines+interleukin-10+(IL-10)+and+IL-1&#946;+in+a+human+tonsil+explant+model.">P2X Antagonists inhibit HIV-1 productive infection and inflammatory cytokines interleukin-10 (IL-10) and IL-1&#946; in a human tonsil explant model.</a> Journal of Virology. 93 (1), 01186 (2019).</li><li>Sorrell, M. E., Hauser, K. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P2X-selective+purinergic+antagonists+are+strong+inhibitors+of+HIV-1+fusion+during+both+cell-to-cell+and+cell-free+infection.">P2X-selective purinergic antagonists are strong inhibitors of HIV-1 fusion during both cell-to-cell and cell-free infection.</a> Journal of Virology. 88 (19), 11504-11515 (2014).</li><li>Soare, A. Y., 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=P2RX7+at+the+host-pathogen+interface+of+infectious+diseases.">P2RX7 at the host-pathogen interface of infectious diseases.</a> Microbiology and Molecular Biology Reviews. 85 (1), (2021).</li><li>Chen, P., H&#252;bner, W., Spinelli, M. A., Chen, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predominant+mode+of+human+immunodeficiency+virus+transfer+between+T+cells+is+mediated+by+sustained+Env-dependent+neutralization-resistant+virological+synapses.">Predominant mode of human immunodeficiency virus transfer between T cells is mediated by sustained Env-dependent neutralization-resistant virological synapses.</a> Journal of Virology. 81 (22), 12582-12595 (2007).</li><li>Dale, B. 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G., 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=Multiparameter+cell+characterization+using+nanofluidic+technology+facilitates+real-time+phenotypic+and+genotypic+elucidation+of+intratumor+heterogeneity.">Multiparameter cell characterization using nanofluidic technology facilitates real-time phenotypic and genotypic elucidation of intratumor heterogeneity.</a> bioRxiv. , 457010 (2018).</li><li>Le, K., 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=Assuring+clonality+on+the+beacon+digital+cell+line+development+platform.">Assuring clonality on the beacon digital cell line development platform.</a> Biotechnology Journal. 15 (1), 1900247 (2020).</li><li>Mocciaro, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-activated+cell+identification+and+sorting+(LACIS)+for+selection+of+edited+clones+on+a+nanofluidic+device.">Light-activated cell identification and sorting (LACIS) for selection of edited clones on a nanofluidic device.</a> Communications in Biology. 1, 41 (2018).</li><li>Winters, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+single+B+cell+antibody+discovery+using+nanopens+and+structured+light.">Rapid single B cell antibody discovery using nanopens and structured light.</a> MAbs. 11 (6), 1025-1035 (2019).</li><li>Wu, N., Nishioka, W. K., Derecki, N. C., Maher, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput-compatible+assays+using+a+genetically-encoded+calcium+indicator.">High-throughput-compatible assays using a genetically-encoded calcium indicator.</a> Science Reports. 9 (1), 12692 (2019).</li><li>Esposito, A. 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=A+high-throughput+cre-lox+activated+viral+membrane+fusion+assay+to+identify+inhibitors+of+HIV-1+viral+membrane+fusion.">A high-throughput cre-lox activated viral membrane fusion assay to identify inhibitors of HIV-1 viral membrane fusion.</a> Journal of Visualized Experiments. (138), e58074 (2018).</li><li>Soare, A. Y., 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=P2X1+selective+antagonists+block+HIV-1+infection+through+inhibition+of+envelope+conformation-dependent+fusion.">P2X1 selective antagonists block HIV-1 infection through inhibition of envelope conformation-dependent fusion.</a> Journal of Virology. 94 (6), 01622 (2020).</li></ol>

R.P.S. is an employee of Sema4. The other authors have no conflicts of interest to disclose.

The described methodology to study the relationship between calcium influx and HIV-1 productive infection in single cells can be adapted to study intracellular calcium kinetics in response to other agonists or antagonists of interest. The preparation of cells for imaging is simple, minimally time-intensive, and reagents are available in convenient kits from widely used manufacturers. Fluo-4-based calcium measurement is well-described in the literature, and HIV-1 can easily be substituted for other viruses or compounds of...

Chronic inflammation is a leading cause of HIV-associated early morbidities and mortality1,2,3. There are multiple mechanisms whereby HIV can activate inflammatory signaling, and recent evidence suggests a role for the P2X receptors in HIV entry which are calcium-gating adenosine triphosphate (ATP) receptors3,4,5,6,7,8,9,10. The P2X subtype of purinergic receptors (P2XR) may be important facilitators of this inflammation. However, the molecular mechanisms of HIV-P2XR interactions are largely unknown and may impact early and late HIV-1 viral life cycle steps. Defining the pathways and kinetics driving HIV-associated chronic inflammation is critical to advancing treatment options for people with HIV.
To assess whether HIV-1 directly agonizes P2X receptors, P2XR activation and HIV-1 infection must be measured in parallel. Assays of P2XR activity and HIV-1 infection have been independently established: Cellular calcium influx is an indicator of P2XR activation, and HIV-1 productive infection can be quantified by RNA abundance. Fluorescence detection of calcium influx is possible with the Fluo-4 calcium-sensitive dye, and HIV-1 infection can be visualized with the mCherry fluorescent reporter virus HIV-NLCI11,12,13,14,15.
Because these indicators of P2X activation (acute cellular calcium influx) and HIV-1 infection (HIV-1 RNA synthesis) occur on different timescales (minutes versus days), there lacks a high-throughput method that allows for paired analysis of P2XR activation and HIV-1 infection. Standard high-throughput experimental techniques, such as flow cytometry, allow for the population analysis but cannot assess the relationship between acute and longitudinal events in single cells. Alternatively, single-cell imaging with standard fluorescence microscopy is low-throughput. These experimental limitations present a need for novel, high-throughput techniques to measure associations between acute and longitudinal cellular events directly.
An optofluidic system described is a novel platform capable of single-cell sorting and isolation, culturing, imaging, and software automation16,17,18,19. This system presents an integrated, high-throughput alternative to the limitations of traditional imaging methods. The Beacon platform consists of a carbon dioxide (CO2) and temperature-controlled incubator that supports cells contained on a chip. The chip possesses photosensitive transistors that generate an electrical gradient in response to targeted light. This resulting dielectrophoretic force is used to move individual cells across the nanofluidic chip to the desired regions. Cells are sorted into pens on the chip, which provide a barrier to isolate individual cells physically. Continuous laminar flow of growth media throughout the chip prevents cell migration from the pens while allowing for small-particle diffusion of nutrients and experiment-specific reagents. A fluorescence microscope sits above the chip. Software automation is used to capture images of the chip at the user-specified time point.
All cell characterization was performed using an optofluidic system for single-cell selection and manipulation. This system consists of integrated mechanical, microfluidic, and optical components that enable single-cell manipulation, assay, culture, and imaging. Cells are loaded and cultured on the disposable nanofluidic device consisting of 3,500 individual chambers (pens), each capable of holding sub-nanoliter volume. Cells can be positioned within pens using light-induced dielectrophoretic &#34;cages&#34; and cultured under temperature- and CO2-controlled conditions. The microfluidics permit perfusion of media or buffers on the chip for cell culture or drug treatment. An actuated needle allows for the import and export of cells from incubated and shuttered well plates.&#160;The chip area can be imaged at 4x or 10x magnification in brightfield and fluorescent channels (including DAPI, FITC, TRed, or Cy5) to characterize cellular phenotypes or functional analysis. The entire system is automated using software that can be used for predesigned workflows or custom experiments.
Relationships between HIV-1 infection and P2XR have been studied, but a high-throughput procedure to directly characterize these interactions in parallel has not been reported. Here, the authors describe a methodology to study HIV-P2XR interactions through tracking acute calcium influx and subsequent HIV-1 productive infection at the single-cell level. Notably, this establishes a novel tool that allows for direct, high-throughput, longitudinal measurement of multiple targets in single cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;Beacon Optofluidic System</td>
			<td>Berkeley Lights</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;FIJI</td>
			<td colspan="2">Open-source software</td>
			<td>PMID: 22743772, 22930834</td>
		</tr>
		<tr>
			<td>&#160;Fluo-4 Calcium Imaging Kit</td>
			<td>Thermo Fisher</td>
			<td>&#160;F10489</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;HIV-1 NLCINL4-3</td>
			<td colspan="2">Laboratory of Benjamin Chen</td>
			<td>PMID: 28148796</td>
		</tr>
		<tr>
			<td>&#160;Hyclone Pennecillin Streptomycin solution</td>
			<td colspan="2">GE Healthcare Life sciences</td>
			<td>&#160;SV30010</td>
		</tr>
		<tr>
			<td>&#160;MT-4 cells</td>
			<td>NIH AIDS Reagent</td>
			<td></td>
			<td>&#160;ARP-120</td>
		</tr>
		<tr>
			<td>&#160;OptoSelect 3500 chip</td>
			<td>Berkeley Lights</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Pipettor Tips</td>
			<td>Denville Scientific</td>
			<td>&#160;P3020-CPS</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Prism 9.0.0</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;RPMI-1640 Medium</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Fisher Brand</td>
			<td>&#160;13-678-11E</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Hood</td>
			<td>Various models</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flasks</td>
			<td>Corning</td>
			<td>3073</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell characterization of calcium influx and hiv-1 infection using a multiparameter optofluidic platform

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Figure 1&#160;illustrates the format of the chip and raw imaging data acquired through the fluorescence microscope. The identification and clustering of uninfected and infected cells via mCherry measurement are shown in Figure 2. These clusters are analyzed for calcium influx kinetics in Figure 3, which demonstrates early calcium influx in HIV-infected cells. Figure 4 shows a significant positive correl...

Watch this Scientific Journal Video about Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform at JoVE.com

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience ...

This protocol can capture real-time kinetics on a single-cell level. We can measure individual cell parameters in response to HIV infection and associate early signaling events with delayed steps of the viral lifecycle. This is an integrated scalable alternative to traditional imaging methods using a novel optofluidic platform for single-cell sorting, culturing, imaging, and software automation.This is the first established method for nanofluidic, high-throughput, longitudinal single-cell culture and imaging. This technique can be broadly adopted to study cellular signaling kinetics and dynamic molecular interactions in a variety of disease states. Visual demonstration of this method is important to convey how the experiment is set up, how cells are optically sorted into pens, and how to set up infusions through the chip.To begin, prepare fresh Fluo-4 AM loading solution by adding 25 microliters of 100X concentrated detergent solvent and 2.5 microliters of 1, 000X Fluo-4 AM to a 1.5 milliliter tube, then vortex to mix. Pipette 2.5 milliliters of culture media into the loading solution and invert to mix. Centrifuge two million MT-4 cells at 500 times G for three minutes, then remove media and resuspend the pellet in two milliliters of the prepared Fluo-4 AM loading solution.Pipette re-suspended cells into a 35 millimeter Petri dish. Incubate at 37 degrees Celsius for 15 to 30 minutes, then incubate for an additional 15 to 30 minutes at room temperature. Transfer the cell suspension to a centrifuge tube and centrifuge the cells at 500 times G for three minutes, then remove the supernatant.Resuspend the cell pellet by pipetting in one milliliter of the culture medium and centrifuge at 500 times G for three minutes to wash. Resuspend the cell pellet by pipetting in the culture medium at a concentration of two million cells per milliliter for at least 50 microliters. To prepare a chip with the wetting solution, which facilitates cell penning, load centrifuge tubes containing two milliliters of wetting solution and 50 milliliters of deionized water onto the instrument with a new optofluidic chip and run the wet chip function which will flood the chip with the wetting solution.Incubate the chip at 50 degrees Celsius and flush the chip with water three times. Once water flushing is done, flush the chip with three cycles of 250 microliters of culture media. Supplement the cell suspension from the previous step with one per 100 parts F127 detergent solute before the loading to reduce the likelihood of cells sticking to the chip channels.Use the instrument export needle to import cells from a 1.5 milliliter centrifuge tube with the load operation and small volume import for a five microliter cell package volume. Pen cells using an optimized optoelectronic positioning or OEP voltage of 4.3 volts and five micrometers per second cage speed by using the auto-pen function, which will auto-detect single cells, surround them with an OEP cage, and move them into a nearby pen. If cells of interest remain following auto-penning, use the manual pen function to select target cells and a destination pen.Once penning is complete, flush the chip with three cycles of 250 microliters of culture media to clear any remaining unpenned cells from the chip. After penning cells, obtain fluorescent images of cells in FITC, Texas Red and DAPI channels to measure the baseline Fluo-4, mCherry, and autofluorescence. Infuse HIV-1 into the microchip at a concentration of 13 nanograms of HIV-1 NLCI per two million cells by slowly pipetting the suspension into the chip through the export needle.Immediately after HIV-1 addition, repeatedly obtain images in the FITC and DAPI channels over a 10-minute time course. Obtain images in the Texas Red and DAPI channels at one, two, three, and four days post-infection. This methodology was used for the identification and clustering of HIV infected and uninfected cells via mCherry measurement.Cells with a change in mCherry signal greater or lower than 40, 000 mean fluorescent intensity were clustered into the mCherry high or mCherry low population respectively. These clusters were analyzed for calcium influx kinetics by measuring intracellular calcium using Fluo-4 fluorescence repeatedly over a nine-minute time course, which demonstrated early calcium influx in HIV-infected cells. A significant positive correlation between calcium influx and mCherry florescence was observed.While attempting this procedure, it's important to remember to start with the cells within the exponential growth range for both virus production and infection because overgrown cells will result in a dramatic decrease in signal from this assay. Working with HIV-1 can be hazardous. Therefore, BSL-2 practices as indicated in the OSHA Bloodborne Pathogen Standard should always be taken while performing this procedure.This technique is exciting because it will allow us to study single-cell phenotypic or transcriptional changes under controlled conditions. This has broad potential for correlating the impact of stimuli on molecular pathways in many cell types.

single-cell-characterization-calcium-influx-hiv-1-infection-using

Research

JoVE Journal

Immunology and Infection

3.0K Görüntüleme.  Icahn School of Medicine at Mount Sinai. Bu protokol tek hücreli düzeyde gerçek zamanlı kinetik yakalayabilir. HIV enfeksiyonuna yanıt olarak bireysel hücre parametrelerini ölçebilir ve erken sinyal olaylarını viral yaşam döngüsünün gecikmiş adımlarıyla ilişkilendirebiliriz. Bu, tek hücreli sıralama, kültleştirme, görüntüleme ve yazılım otomasyonu için yeni bir optofluidik platform kullanan geleneksel görüntüleme yöntemlerine entegre ölçeklenebilir bir alternatiftir.Bu nanoakışkan, yüksek...