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.
