3.4 Sorting
of Chlamydia-Specific
Cells
1. The sorter should be fitted with a 70 μm nozzle for optimal
sorting and viability of human immune cells.
2. Set the compensation values for the sort using single-stained
compensation beads for each of the fluorescent antibodies and
the viability dye.
3. Use a fluorescence minus one (FMO) control stained with
viability dye and fluorescent antibodies but lacking the
DiO-labeled EBs to set the gates for the sort. Acquire at least
10
6 events on the cell sorter from the FMO sample (see Note
8).
4. To gate for the cells of interest (see Fig. 2), first create a plot
examining FSC-A and SSC-A. Adjust the threshold value of
FSC-A to remove small debris from the analysis, and set a gate
around the lymphocyte population.
5. From the lymphocytes, create a plot with FSC-A and FSC-H in
order to define singlets and remove aggregates from the
analysis.
6. From the singlet gate, create a plot of CD3/14 and CD19. Set
a gate around the CD19
+ CD3/14
À population to identify the
B cells.
7. Within the B cell population, create a plot of CD27 and CD38.
Set a gate around the CD27
+ CD38
À population to identify
memory B cells (MBCs).
8. Within the MBC population, create a plot of IgG and IgA. Set a
gate around the IgG
+ IgA
À population (see Note 9).
9. To remove any mistakenly-gated unswitched memory B cells,
create a plot within the IgG
+ IgA
À population of IgM and IgD.
Set a gate around the IgM
À IgD
À population to identify the
IgG
+ MBCs.
10. Within the IgG
+ MBC population, create a plot to show DiO.
Set the DiO
+ gate immediately above the negative population
shown in the FMO control.
11. Using these gates, sort the DiO
+ IgG
+ MBC population into a
tube or plate containing a buffer dependent on downstream
analysis, such as culture media or RNA preservation buffer (see
Note 10).
4 Notes
1. Dilution of blood is needed to reduce the density of the sample
so as to not “overload” the Histopaque layer and reduce
PBMC recovery. If the blood sample has been kept for several
hours before PBMC isolation, it may be necessary to increase
the dilution with PBS to 2:1 to counter potential aggregation.
Isolation of Chlamydia-Specific B Cells
25
of Chlamydia-Specific
Cells
1. The sorter should be fitted with a 70 μm nozzle for optimal
sorting and viability of human immune cells.
2. Set the compensation values for the sort using single-stained
compensation beads for each of the fluorescent antibodies and
the viability dye.
3. Use a fluorescence minus one (FMO) control stained with
viability dye and fluorescent antibodies but lacking the
DiO-labeled EBs to set the gates for the sort. Acquire at least
10
6 events on the cell sorter from the FMO sample (see Note
8).
4. To gate for the cells of interest (see Fig. 2), first create a plot
examining FSC-A and SSC-A. Adjust the threshold value of
FSC-A to remove small debris from the analysis, and set a gate
around the lymphocyte population.
5. From the lymphocytes, create a plot with FSC-A and FSC-H in
order to define singlets and remove aggregates from the
analysis.
6. From the singlet gate, create a plot of CD3/14 and CD19. Set
a gate around the CD19
+ CD3/14
À population to identify the
B cells.
7. Within the B cell population, create a plot of CD27 and CD38.
Set a gate around the CD27
+ CD38
À population to identify
memory B cells (MBCs).
8. Within the MBC population, create a plot of IgG and IgA. Set a
gate around the IgG
+ IgA
À population (see Note 9).
9. To remove any mistakenly-gated unswitched memory B cells,
create a plot within the IgG
+ IgA
À population of IgM and IgD.
Set a gate around the IgM
À IgD
À population to identify the
IgG
+ MBCs.
10. Within the IgG
+ MBC population, create a plot to show DiO.
Set the DiO
+ gate immediately above the negative population
shown in the FMO control.
11. Using these gates, sort the DiO
+ IgG
+ MBC population into a
tube or plate containing a buffer dependent on downstream
analysis, such as culture media or RNA preservation buffer (see
Note 10).
4 Notes
1. Dilution of blood is needed to reduce the density of the sample
so as to not “overload” the Histopaque layer and reduce
PBMC recovery. If the blood sample has been kept for several
hours before PBMC isolation, it may be necessary to increase
the dilution with PBS to 2:1 to counter potential aggregation.
Isolation of Chlamydia-Specific B Cells
25