8. Pour off the supernatant and resuspend the cells in 1 mL of 1%
PBS/BSA. Count the cells using any preferred method.
9. Centrifuge the cells for 5 min at 350 Â g at room temperature.
Remove the supernatant and resuspend in freezing medium to
a concentration of 1 Â 10
7 cells/mL.
10. Aliquot the cell suspension into cryovials at a volume of 1 mL
per tube. Place the tubes in a cell freezing chamber and transfer
the chamber to a À80
C freezer. After 24 h, the tubes can be
moved to a À150
C freezer or a liquid nitrogen storage vessel.
This allows samples to be stored and shipped prior to downstream analysis.
3.2 Preparation
of Labeled EBs
Elementary bodies (EBs) of C. trachomatis should be purified and
quantified (see Note 2) using a preferred in-house method, which
will not be covered here. For the purpose of this protocol, aliquots
containing 10
7 purified EBs in SPG buffer should be prepared from
a stock and stored at À80
C. Labeled EBs should be prepared fresh
from these aliquots and used for cell staining on the same day.
1. Thaw an aliquot of 10
7 EBs on wet ice for 5–10 min or until
liquid.
2. Add 1 mL of PBS to the EB sample and transfer to a 1.5 mL
microcentrifuge tube. Centrifuge for 20 min at >20,000 Â g at
4
C (see Notes 3 and 4).
3. Carefully aspirate the supernatant using a 200 μL pipette without disturbing the EB pellet area.
4. Add 500 μL of prepared fixation buffer to the tube and vortex
to resuspend the EBs. Incubate for 20 min at room
temperature.
5. Add 500 μL of PBS to the tube and vortex. Centrifuge for
20 min at >20,000 Â g at 4
C.
6. Carefully aspirate the supernatant using a 200 μL pipette without disturbing the EB pellet.
7. Add 500 μL of PBS containing 1 μL of Vybrant DiO staining
solution to the EBs. Vortex to resuspend them and incubate for
20 min at 37
C protected from light.
8. Add 500 μL of 1% PBS/BSA and transfer the EB suspension to
a new 1.5 mL microcentrifuge tube (see Note 5).
9. Centrifuge for 20 min at >20,000 Â g at 4
C. Carefully
aspirate the supernatant using a 200 μL pipette without disturbing the EB pellet.
10. Add 100 μL of 1% PBS/BSA to the tube and vortex to resuspend the EBs. Keep this suspension on ice protected from
light.
Isolation of Chlamydia-Specific B Cells
23
PBS/BSA. Count the cells using any preferred method.
9. Centrifuge the cells for 5 min at 350 Â g at room temperature.
Remove the supernatant and resuspend in freezing medium to
a concentration of 1 Â 10
7 cells/mL.
10. Aliquot the cell suspension into cryovials at a volume of 1 mL
per tube. Place the tubes in a cell freezing chamber and transfer
the chamber to a À80
C freezer. After 24 h, the tubes can be
moved to a À150
C freezer or a liquid nitrogen storage vessel.
This allows samples to be stored and shipped prior to downstream analysis.
3.2 Preparation
of Labeled EBs
Elementary bodies (EBs) of C. trachomatis should be purified and
quantified (see Note 2) using a preferred in-house method, which
will not be covered here. For the purpose of this protocol, aliquots
containing 10
7 purified EBs in SPG buffer should be prepared from
a stock and stored at À80
C. Labeled EBs should be prepared fresh
from these aliquots and used for cell staining on the same day.
1. Thaw an aliquot of 10
7 EBs on wet ice for 5–10 min or until
liquid.
2. Add 1 mL of PBS to the EB sample and transfer to a 1.5 mL
microcentrifuge tube. Centrifuge for 20 min at >20,000 Â g at
4
C (see Notes 3 and 4).
3. Carefully aspirate the supernatant using a 200 μL pipette without disturbing the EB pellet area.
4. Add 500 μL of prepared fixation buffer to the tube and vortex
to resuspend the EBs. Incubate for 20 min at room
temperature.
5. Add 500 μL of PBS to the tube and vortex. Centrifuge for
20 min at >20,000 Â g at 4
C.
6. Carefully aspirate the supernatant using a 200 μL pipette without disturbing the EB pellet.
7. Add 500 μL of PBS containing 1 μL of Vybrant DiO staining
solution to the EBs. Vortex to resuspend them and incubate for
20 min at 37
C protected from light.
8. Add 500 μL of 1% PBS/BSA and transfer the EB suspension to
a new 1.5 mL microcentrifuge tube (see Note 5).
9. Centrifuge for 20 min at >20,000 Â g at 4
C. Carefully
aspirate the supernatant using a 200 μL pipette without disturbing the EB pellet.
10. Add 100 μL of 1% PBS/BSA to the tube and vortex to resuspend the EBs. Keep this suspension on ice protected from
light.
Isolation of Chlamydia-Specific B Cells
23