3.6 Flow Cytometer
Shutdown
1. Shut down the flow cytometer according to manufacturer’s
protocol. The process including running decontamination,
cleaning, and deionized water solutions takes approximately
30 min.
2. The flow cytometer automatically powers off after an internal
washing cycle.
4 Notes
1. If possible, use sterile material to avoid analysis by the flow
cytometer of debris or contaminants which could generate
important background noise.
2. Other combination of viability fluorescent dyes may be used
with the following specifications: the first dye should stain all
cells (e.g., other SYTO stains, SYBR-II, DAPI, or thiazole
orange) or only living cells (e.g., CFDA), while the second
should only mark dying and dead cells (e.g., TO-PRO-3
iodide). Their excitation wavelengths should correspond to a
flow cytometer laser emission wavelength, and their emission
wavelengths to two different flow cytometer detectors. Avoid
spillover between the two dyes’ emission spectra as it would
require implementing flow cytometer compensation.
3. Other flow cytometers can be used. Check if laser emission and
detectors’ wavelengths correspond to the desired fluorochromes. Here, we describe the use of the benchtop BD
Accuri™ C6 flow cytometer.
4. These dilution factors were determined to obtain a speed of
analysis compatible with our flow cytometer performance.
According to Accuri™ C6 manufacturer, the system can
accommodate an upper limit of 10,000 events per second,
but it is recommended to acquire samples at a rate of 2500
events per second or less to ensure the best data resolution. We
experimentally determined that enumeration is statistically linear up to 7000 events per second corresponding to 30,000
events per microliter [9], but we routinely perform dilutions to
reach a speed of analysis of approximately 250 events per
second or 1000 events per microliter for maximal data
resolution.
5. Perform the dilution first and then the staining. Staining will
not be optimal otherwise.
6. Do not incubate bacteria with PI for an extended period of
time (>10 min) since the dye is toxic and slowly kills bacteria. It
would result in an overestimation of dead cells.
7. Dyes are sensitive to light. Although 3 min of incubation
exposed to light will not result in any significant degradation
38
Ce ´ lia Fontana and Jean Reyes
Shutdown
1. Shut down the flow cytometer according to manufacturer’s
protocol. The process including running decontamination,
cleaning, and deionized water solutions takes approximately
30 min.
2. The flow cytometer automatically powers off after an internal
washing cycle.
4 Notes
1. If possible, use sterile material to avoid analysis by the flow
cytometer of debris or contaminants which could generate
important background noise.
2. Other combination of viability fluorescent dyes may be used
with the following specifications: the first dye should stain all
cells (e.g., other SYTO stains, SYBR-II, DAPI, or thiazole
orange) or only living cells (e.g., CFDA), while the second
should only mark dying and dead cells (e.g., TO-PRO-3
iodide). Their excitation wavelengths should correspond to a
flow cytometer laser emission wavelength, and their emission
wavelengths to two different flow cytometer detectors. Avoid
spillover between the two dyes’ emission spectra as it would
require implementing flow cytometer compensation.
3. Other flow cytometers can be used. Check if laser emission and
detectors’ wavelengths correspond to the desired fluorochromes. Here, we describe the use of the benchtop BD
Accuri™ C6 flow cytometer.
4. These dilution factors were determined to obtain a speed of
analysis compatible with our flow cytometer performance.
According to Accuri™ C6 manufacturer, the system can
accommodate an upper limit of 10,000 events per second,
but it is recommended to acquire samples at a rate of 2500
events per second or less to ensure the best data resolution. We
experimentally determined that enumeration is statistically linear up to 7000 events per second corresponding to 30,000
events per microliter [9], but we routinely perform dilutions to
reach a speed of analysis of approximately 250 events per
second or 1000 events per microliter for maximal data
resolution.
5. Perform the dilution first and then the staining. Staining will
not be optimal otherwise.
6. Do not incubate bacteria with PI for an extended period of
time (>10 min) since the dye is toxic and slowly kills bacteria. It
would result in an overestimation of dead cells.
7. Dyes are sensitive to light. Although 3 min of incubation
exposed to light will not result in any significant degradation
38
Ce ´ lia Fontana and Jean Reyes