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

Summary

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.

Abstract

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.

Introduction

Chronic inflammation is a leading cause of HIV-associated early morbidities and mortality^1,2,3. 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) receptors^{3,4,5,6,7,8,9,10}. 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-NLCI^{11,12,13,14,15}.

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 automation^16,17,18,19. 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 "cages" 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. 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.

Protocol

1. Preparation of cells for imaging

1. Prepare fresh Fluo-4 AM loading solution: Add 25 µL of 100x concentrated detergent solvent, then add 2.5 µL of Fluo-4 AM 1000x to a 1.5 mL tube. Vortex to mix.
2. Pipette 2.5 mL of culture media into the loading solution and invert to mix. Protect from light.
3. Centrifuge 2 x 10⁶ MT-4 cells at 500 x g for 3 min.
4. Remove media and resuspend the pellet in 2 mL of the prepared Fluo-4 AM loading solution. Protect from light.
5. Pipette resuspended cells into a 35 mm Petri dish. Incubate at 37 °C for 15-30 min, then incubate for an additional 15-30 min at room temperature.
6. Transfer the cell suspension to a centrifuge tube. Centrifuge cells at 500 x g for 3 min and remove the supernatant consisting of the loading solution.
7. Resuspend the cell pellet by pipetting in 1 mL of the culture medium and centrifuge at 500 x g for 3 min to wash.
8. Resuspend the cell pellet by pipetting in the culture medium at a concentration of 2 x 10⁶ cells/mL (minimum 50 µL). Protect from light.

2. Optofluidic system preparation, cell loading, and cell penning

1. Prepare a chip with the wetting solution, which facilitates cell penning.
   1. To do this, load centrifuge tubes containing 2 mL of wetting solution and 50 mL of DI water onto the instrument with a new optofluidic chip and run the Wet Chip function. This function will automatically flood the chip with the wetting solution through the system fluidics, incubate the chip at 50, °C and flush the chip with water 3 times.
   2. Once water flushing is done, flush the chip with 3 cycles of 250 µL of culture media.
2. Supplement the cell suspension from step 1.8 with 1:100 F-127 detergent solute before the loading to reduce the likelihood of cells sticking to the chip channels.
3. Use the instrument export needle to import cells from a 1.5 mL centrifuge tube using the Load operation and Small Volume Import for a 5 µL cell package volume.
4. Pen cells using an optimized optoelectronic positioning (OEP) voltage of 4.3 V and 5 µm/s cage speed. Penning occurs using OEP. Accomplish penning using the Autopen function, which will autodetect 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 (with a mouse click) and a destination pen (with a subsequent mouse click).
5. Once penning is complete (either when the Autopen function completes or when the desired number of penned cells has been attained), flush the chip with 3 cycles of 250 µL of culture media to clear any remaining unpenned cell from the chip.

3. Single-cell infection and functional imaging

1. After penning cells, obtain fluorescent images of cells in FITC, Texas Red, and DAPI channels to measure the baseline Fluo-4, mCherry, and autofluorescence.
2. Infuse HIV-1 into the microchip at a concentration of 13 ng of HIV-1 NL-CI per 2 x 10⁶ cells by slowly pipetting the suspension into the chip through the export needle.
3. Immediately after HIV-1 addition, repeatedly obtain images in the FITC and DAPI channels over a 10-min time course.
4. Obtain images in the Texas Red and DAPI channels at 1-, 2-, 3-, and 4-days post-infection (DPI).

4. Analysis of single-cell functional imaging data with FIJI software

1. At each time point in each imaging channel of interest, use the FIJI Point Selection tool and the Measure function to measure each cell's Fluo-4, mCherry, and autofluorescence signals. Simultaneously measure the background fluorescence by calculating the mean fluorescence intensity (MFI) of the pen and chip containing each cell.
2. Control for the background fluorescence and autofluorescence by using the Measure function to find the MFI in a Region Selection and subtracting this value from the cell fluorescence value.
3. Normalize the background fluorescence of each chip image: Measure the MFI of the chip in each field. Then subtract the MFI of the chip with the least background fluorescence from all other chip MFI measurements.
4. Normalize the background fluorescence of each cell-containing pen: measure the MFI of each cell's pen in each field of view. Then subtract this value from the cell's raw MFI for each field.
5. Control for the cell autofluorescence: Subtract the day 0 and day 4 DAPI MFI of each cell from the day 0 and day 4 mCherry MFIs.

Results

Figure 1 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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Beacon Optofluidic System	Berkeley Lights
Fetal Bovine Serum	Gibco
FIJI	Open-source software		PMID: 22743772, 22930834
Fluo-4 Calcium Imaging Kit	Thermo Fisher	F10489
HIV-1 NLCINL4-3	Laboratory of Benjamin Chen		PMID: 28148796
Hyclone Pennecillin Streptomycin solution	GE Healthcare Life sciences		SV30010
MT-4 cells	NIH AIDS Reagent		ARP-120
OptoSelect 3500 chip	Berkeley Lights
Pipettor Tips	Denville Scientific	P3020-CPS
Prism 9.0.0	GraphPad
RPMI-1640 Medium	Sigma-Aldrich	R8758
Serological Pipettes	Fisher Brand	13-678-11E
Tissue Culture Hood	Various models
T75 flasks	Corning	3073