The overall goal of the following experiment is to utilize fluorescent dyes to monitor changes in membrane dynamics and intracellular ion concentrations in a bacterial system. This is achieved by first incubating bacteria during their growth phase with the fluor of interest until it has equilibrated across their membranes. Next, the bacteria are treated with the agent of choice and their fluorescence is monitored.
The results can show changes in membrane depolarization, membrane integrity, and intracellular ion concentrations based on the observed changes in fluorescence of the indicator dyes. The main advantage of this technique over conventional electrophysiology techniques like patch clamping that is used in eukaryotic systems is that changes in membrane dynamics and intracellular ion concentrations can now be monitored in bacteria using fluorescent. Generally individuals new to this method will struggle because it takes some effort to optimize the dye concentrations.
Loading conditions and detection settings Begin with bacterial cells. Just pelleted in wash solution first resuspend the pellet in at least one milliliter of PBS for 100 million CFU per milliliter, which is enough for five samples. Transfer one milliliter of the washed cells to a micro centrifuge tube and add 25 microliters of a one molar filter.
Sterilized stock solution of glucose then incubate the cells in a 37 degrees Celsius heating block for 15 minutes. The added glucose may vary based on the experiment. After 15 minutes, add five microliters of the 50 micromolar dieback, and 10 microliters of PI solution.
Mix the cells thoroughly by pipetting them up and down several times. Then divide this cell suspension into five sample wells of a clear 96 well plate. Load the plate into a prewarm plate reader set for dieback NP detection.
Now monitor the dieback equilibration over the cell membranes by taking measurements once per minute over 30 to 40 minutes until the reading stabilize to their pretreatment value. Then eject the plate and add the experimental agent of choice. Immediately return the plate to the reader and continue monitoring dieback and PI fluorescence for the desired length of time.
When preparing the loading buffer for this procedure, be sure to vortex it briefly before adding PBS and Probenecid. After adding those solutions, vortex it again and wrap it in foil to one milliliter of two x cell suspension. Add one milliliter of two x loading buffer.
This is enough for 10 samples. Incubate the mix in a 37 degree Celsius water bath for 75 minutes, protected from light Throughout all of these assays. The concentration of the dye and the incubation time may need to be adjusted to optimize the assay and the baseline fluorescence detection level, depending on the specific bacterial species and other experimental system variables.
After the incubation, wash the cells three times for each wash. Pellet the cells at 2, 400 Gs for 10 minutes and resuspend them in two milliliters of PBS containing one x pronic. After the three washes, incubate the cells at 37 degrees Celsius for another 30 minutes.
This enables estro inside the bacteria to hydrolyze the ester groups of the dye, rendering the dye active within the cell. Next pellet, the cells remove the snat and resus suspend them in two milliliters of PBS containing one x Probenecid. If needed, glucose can now be added back to reenergize the bacteria load 200 microliters of the cells into one well of a 96 well plate for each sample and load the plate into a prewarm reader in one sample.
Well first establish a baseline reading at the proper wavelengths and at the rate of one reading per second for one minute. Then remove the plate and add about five microliters of the treatment of choice. To that.
Well quickly pipette up and down a few times to mix in the treatment and immediately return the plate to the reader. Continue taking measurements for the desired time. To analyze the data, calculate the ratio of the fluorescent values for each time point by dividing value, one by value two, and plot the ratio values on a graph.
In preparation, create a range of five or six high potassium buffers with pH values from 6.5 to 8.0. As in the previous procedure, use a vortex to prepare the two x loading buffer from a mid log phase culture wash and concentrate the cells. Then replace the SNAT with two milliliters of BS To make a four x cell suspension.
Combine one milliliter of the four x cell suspension with one milliliter of the two x loading buffer. Now incubate the cells in a 30 degree Celsius water bath for 40 minutes. Protect it from light.
Now wash the cells three times. Pellet the cells as shown previously. Add back four milliliters of PBS containing two x prop Benin, and one millimolar glucose and repeat the centrifugation after the three washes.
Incubate the cells at 37 degrees Celsius for another five minutes. To determine the background fluorescence filter, sterilize about 500 microliters of the energized bacterial suspension to remove the bacteria. Then transfer 200 microliters of the filtrate to a 96 Well plate and measure the absorption from that well for a minute.
To obtain an in vivo calibration curve, spin down 500 microliter ALI watts of the loaded and energized cells and resus. Suspend them in a series of high potassium buffers of different pH to each sample, add 20 micromolar of risin. This equilibrates the intracellular pH of the cells to the pH of the surrounding buffer.
Then incubate the samples at 37 degrees Celsius for five minutes. After the incubation, transfer the samples to the 96 Well plate in the plate reader. Measure the fluorescence of each sample for a few minutes to establish a study reading for each.Well.
To analyze this data, subtract the background fluorescence values, and then calculate the ratio of the fluorescence values by dividing value. One by value two, plot the ratio values for each pH buffer to create the calibration curve to measure changes in intracellular pH. Load 200 microliter samples of the energized cells into a plate and measure the fluorescence every five seconds for five minutes.
Simultaneously, add 10 micromolar CCCP to one sample while adding the agents of interest to the other samples. The CCCP is a time aligned positive control for decreasing intracellular pH. Immediately return the plate to the reader and continue measuring fluorescence every five seconds for 10 minutes.
As an added control, add 20 micro molars NI garrison to all of the samples to equilibrate the pH of the bacteria to that of the buffer for another five minutes. When exploring membrane polarity, the bacteria need to be incubated with die back for about 40 minutes to allow the dye to equilibrate over the membrane. This is indicated by the steady decrease in fluorescence that levels off.
PI does not require equilibration, but along with dieback, it is helpful for monitoring membrane rupture. Concurrently with polarity, depolarization and rupture of the bacteria is indicated by a rise in fluorescent intensity of both dyes for fewer 2:00 AM the ratio of two fluorescent signals is calculated and an increase or decrease in this ratio corresponds to the intracellular calcium. For PBFI am intracellular potassium is measured by fluorescence ratios.
Intracellular hydrogen is measured using fluorescence ratios of B-C-E-C-F am. Upon addition of the calcium iono four ion mycin calcium flows into the cell causing an increase of the fluorescence ratio. Addition of valmy has the opposite effect causing potassium to flow out of the cell, which is indicated by a decrease in the fluorescence ratio.
B-C-E-C-F allows for the measurement of intracellular pH first calibration curves are made in various buffers of known pH. A decrease in the fluorescence ratio of the dye corresponds to a decrease in pH. This is seen with the addition of proton of force CCCP, the black line, and after the addition of a proton motive force dissipator the blue line.
The second arrow shows addition of nissin, which equilibrates intracellular and extracellular hydrogen concentrations. After watching this video, you should have a good understanding of how to measure membrane depolarization, membrane integrity, and intracellular concentrations of ions in bacterial cells. You should also be able to use this protocol as a starting point to study other properties and bacterial cells.
