We develop high-throughput microphilic IC methods to study cell population diversity and cell-cell interactions. In this context, gel microdroplets enable us to perform useful protocol steps, like lysis-staining DNA manipulation and microscopy in a highly paralyzed manner in trapped cells. Droplet microphilic workflows are known for the ultra-high throughput and versatility, but adoption is often limited by the need for high-end instrumentation and specialized expertise.
Our protocol improves traditional techniques using high-throughput, single-cell analysis with fluorescent staining and an accessible open-source microscopy workflow. It encapsulates cells in gel microdroplets via microfluidics to analyze minicells and microcolonies simultaneously. While bulk and plate cultures lack specificity and resolution, this method gels precise and representative statistics of diverse phenomena.
Our protocols combine droplet microfluidics, fluorescent microscopy and open-source hardware in a label-based method. This approach makes single-cell analysis more flexible and affordable, enabling the scientific community to address complex scientific question regarding microbial communities. To begin, heat ultra-low-gelling-temperature agarose at a concentration of 2%to 90 degrees Celsius in Luria Bertani, or LB broth.
Shake the mixture for 10 minutes in a temperature-controlled shaker, then reduce the temperature of the thermo shaker to 39 degrees Celsius to cool the agarose solution. Place the Escherichia coli suspension tube in the thermo shaker for four minutes to warm it to 39 degrees Celsius. Mix the bacteria suspension and agarose solution in a one-to-one ratio to achieve an agarose concentration of 1%with a cell suspension of 3.1 times 10 to the power of six cells per milliliter.
Prepare the negative control solution for contamination monitoring using LB and agarose suspension in a one-to-one ratio. Obtain the full open-source flow platform, including gas pressure drivers and flow sensors. Include a glass slide and pipette tip heaters in the setup to control the temperature of the agarose cell sample as it enters the chip.
Next, position the microfluidic chip on the strobe-enhanced microscopy stage, ensuring that the droplet generation junction is visible. Set the pipette tip heater and glass slide heater to 40 degrees Celsius using the control software interface. Using a syringe with tubing and a PDMS plug, load 1%agarose cell suspension mix into a 200-microliter pipette tip.
Insert the tip into the tip heater and place it on the inlet of the aqueous phase in the microfluidic chip. Exchange the tip's PDMS seal with the one connected to the flow control system tubing. Next, insert the oil tubing into the second inlet and the outlet tubing end into a waste tube.
Then set the pressure to 80 millibars for the acquiesce and oil phases on the user interface, and start the infusion of the cell suspension and oil. Adjust the oil phase to 320 millibars and the acquiesce phase to 180 millibars. Allow one minute for stabilization of droplet generation.
Once droplet generation stabilizes, transfer the outlet tubing from the waste tube to the collection tube. Continue collecting droplets on ice until the sample reservoir is empty. After collecting, place the tubes at four degrees Celsius for one hour to let the agarose gel inside the droplets.
Incubate the gel microdroplets containing bacteria and the negative control droplets at 37 degrees Celsius for four hours or overnight for colony growth. After incubation, use a three-milliliter syringe with a 21-gauge needle to carefully remove as much oil as possible from the base of the gel microdroplet emulsion. Transfer 50 microliters of the gel microdroplets to a new microtube, and store it at four degrees Celsius for further droplet analysis, add a one-to-one mixture of fluorinated oil with PFO in a volume equal to the remaining emulsion.
Next, add approximately 200 microliters of 0.9%sodium chloride buffer on top of the emulsion. Vortex the mixture, and briefly spin it down in a fixed-speed centrifuge. Carefully remove the oil phase from the bottom of the liquid interface and discard 100 microliters of sodium chloride solution from the top.
Transfer two microliters of gel microdroplets into an imaging chamber slide and add five microliters of fluorinated oil to help form a monolayer of droplets for optimal imaging. On the microscope, activate the white LED matrix illumination from the top for brightfield imaging. Mount the prepared slide.
Focus on the sample to locate a monolayer of droplets and capture a brightfield image. Next, adjust the filter wheel to align with the green wavelength filter. Switch to the 470-nanometer LED for excitation, and without moving the sample, capture a fluorescent image of the colonies.
Transfer two microliters of the propidium iodide-stained micro-gels into an imaging chamber chip and add five microliters of 0.9%sodium chloride solution to form a monolayer of micro-gels. Seal the inlet and outlet of the chip to prevent evaporation during imaging. Adjust the red wavelength interval filter for propidium iodide imaging, and capture the brightfield and fluorescent images.
Cell encapsulation in gel microdroplets was confirmed by brightfield microscopy, which shows uniform droplets with distinct encapsulated cells. Colonies that lost plasmid-encoded fluorescence were identified using fluorescence ratio analysis. Out of 2, 785 colonies analyzed, 100 colonies lost fluorescence, indicating a plasmid loss rate of 3.6%
