2.4 Flow Cytometry
Analysis
1. BD Accuri™ C6 flow cytometer combined with BD Accuri™
C6 software (BD Biosciences): This flow cytometer is composed of a blue laser emitting at 488 nm, a forward scatter
detector (FSC) at 0
, a side scatter detector (SSC) at 90
, and
fluorescence detectors in particular a green fluorescence one,
FL1, detecting at 533 Æ 15 nm and a red one, FL3, detecting
at >670 nm (Fig. 1). An automatic calculation allows the
determination of the absolute count of events per microliter
(see Note 3).
2. 0.22 μm filtered, deionized water.
3. Validation beads supplied by flow cytometer manufacturer.
3 Methods
3.1 Flow Cytometer
Start-Up
1. Launch the BD Accuri™ C6 flow cytometer according to
manufacturer’s protocol. The start-up process including running manufacturer’s validation beads to monitor flow cytometer performance takes approximately 20 min.
3.2 Preparation
of Leptospira Prior
to Flow Cytometry
Analysis
1. Measure culture OD 450 nm to evaluate its density. If no spectrophotometer is available, evaluate the density by observing
the bacteria under dark-field microscopy.
2. For non-detectable OD 450 nm , usually corresponding to culture less concentrated than 10
7
–10
8 leptospires/mL, dilute the
bacteria at 1:10 in saline solution. For 0 < OD 450 nm 0.3,
dilute the bacteria at 1:100 in saline solution (two serial dilutions at 1:10). For OD 450 nm > 0.3, dilute the bacteria at
1:1000 in saline solution (three serial dilutions at 1:10) (see
Note 4).
Fig. 1 Excitation and emission spectra of SYTO
® 9 and PI dyes superimposed with BD Accuri™ C6 laser
emission and detector wavelengths. (Adapted from Fluorescence SpectraViewer (https:/ /www.thermofisher.
com/de/de/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html))
Enumeration of Leptospira Cells
33
Analysis
1. BD Accuri™ C6 flow cytometer combined with BD Accuri™
C6 software (BD Biosciences): This flow cytometer is composed of a blue laser emitting at 488 nm, a forward scatter
detector (FSC) at 0
, a side scatter detector (SSC) at 90
, and
fluorescence detectors in particular a green fluorescence one,
FL1, detecting at 533 Æ 15 nm and a red one, FL3, detecting
at >670 nm (Fig. 1). An automatic calculation allows the
determination of the absolute count of events per microliter
(see Note 3).
2. 0.22 μm filtered, deionized water.
3. Validation beads supplied by flow cytometer manufacturer.
3 Methods
3.1 Flow Cytometer
Start-Up
1. Launch the BD Accuri™ C6 flow cytometer according to
manufacturer’s protocol. The start-up process including running manufacturer’s validation beads to monitor flow cytometer performance takes approximately 20 min.
3.2 Preparation
of Leptospira Prior
to Flow Cytometry
Analysis
1. Measure culture OD 450 nm to evaluate its density. If no spectrophotometer is available, evaluate the density by observing
the bacteria under dark-field microscopy.
2. For non-detectable OD 450 nm , usually corresponding to culture less concentrated than 10
7
–10
8 leptospires/mL, dilute the
bacteria at 1:10 in saline solution. For 0 < OD 450 nm 0.3,
dilute the bacteria at 1:100 in saline solution (two serial dilutions at 1:10). For OD 450 nm > 0.3, dilute the bacteria at
1:1000 in saline solution (three serial dilutions at 1:10) (see
Note 4).
Fig. 1 Excitation and emission spectra of SYTO
® 9 and PI dyes superimposed with BD Accuri™ C6 laser
emission and detector wavelengths. (Adapted from Fluorescence SpectraViewer (https:/ /www.thermofisher.
com/de/de/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html))
Enumeration of Leptospira Cells
33