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,}

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 ....

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