used. However a higher speed rotor with a maximum speed of
>30,000 Â g can also be used for a higher recovery of EBs.
4. Due to the small size of the EBs, a pellet will not be visible with
the numbers used here. Before loading the tube into the centrifuge, use a marker pen to indicate the side of the tube that
will be facing the outside edge of the rotor where the EBs will
pellet.
5. DiO is a hydrophobic dye and requires strong vortexing to mix
into an aqueous solution. It will also adhere to the walls of
microcentrifuge tubes, so after incubation of the EBs with DiO
it is necessary to transfer the EB suspension to a fresh tube to
limit contamination of the labeled EBs with free DiO.
6. Both CD3 and CD14 are used to identify abundant non-B cells
in the PBMC sample and as such can be conjugated to the same
fluorochrome in a “dump” channel.
7. An IgG antibody should be identified that does not bind to the
antigen-binding portion of the IgG molecule and would
inhibit binding of the EB to the B cell.
8. The FMO should be an identically-processed PBMC sample,
thawed and stained concurrently with the experimental sample
(s). However this sample does not need to contain B cells
reactive against C. trachomatis EBs and thus can be isolated
from a control sample, removing the need to use a valuable
experimental sample.
9. C. trachomatis–reactive MBCs can also be identified and sorted
from the IgA
+ population using a similar gating strategy that
includes removal of IgM
+ and IgD
+ MBCs.
10. If C. trachomatis–reactive B cells are to be sorted for the
purpose of cloning and/or sequencing of the Ig mRNA, the
sorting procedure should take place under RNA-sterile
conditions.
Acknowledgments
We would like to thank Prof. Martin Holland and Dr. Harry Pickering of the London School of Hygiene and Tropical Medicine, and
Drs. Frank Follmann, Ida Rosenkrands, and Anja Olsen of the
Statens Serum Institut for providing the purified elementary bodies
used for this work.
References
1. Newman L, Rowley J, Hoorn Vander S et al
(2015) Global estimates of the prevalence and
incidence of four curable sexually transmitted
infections in 2012 based on systematic review
and global reporting. PLoS One 10:1–17
Isolation of Chlamydia-Specific B Cells
27
>30,000 Â g can also be used for a higher recovery of EBs.
4. Due to the small size of the EBs, a pellet will not be visible with
the numbers used here. Before loading the tube into the centrifuge, use a marker pen to indicate the side of the tube that
will be facing the outside edge of the rotor where the EBs will
pellet.
5. DiO is a hydrophobic dye and requires strong vortexing to mix
into an aqueous solution. It will also adhere to the walls of
microcentrifuge tubes, so after incubation of the EBs with DiO
it is necessary to transfer the EB suspension to a fresh tube to
limit contamination of the labeled EBs with free DiO.
6. Both CD3 and CD14 are used to identify abundant non-B cells
in the PBMC sample and as such can be conjugated to the same
fluorochrome in a “dump” channel.
7. An IgG antibody should be identified that does not bind to the
antigen-binding portion of the IgG molecule and would
inhibit binding of the EB to the B cell.
8. The FMO should be an identically-processed PBMC sample,
thawed and stained concurrently with the experimental sample
(s). However this sample does not need to contain B cells
reactive against C. trachomatis EBs and thus can be isolated
from a control sample, removing the need to use a valuable
experimental sample.
9. C. trachomatis–reactive MBCs can also be identified and sorted
from the IgA
+ population using a similar gating strategy that
includes removal of IgM
+ and IgD
+ MBCs.
10. If C. trachomatis–reactive B cells are to be sorted for the
purpose of cloning and/or sequencing of the Ig mRNA, the
sorting procedure should take place under RNA-sterile
conditions.
Acknowledgments
We would like to thank Prof. Martin Holland and Dr. Harry Pickering of the London School of Hygiene and Tropical Medicine, and
Drs. Frank Follmann, Ida Rosenkrands, and Anja Olsen of the
Statens Serum Institut for providing the purified elementary bodies
used for this work.
References
1. Newman L, Rowley J, Hoorn Vander S et al
(2015) Global estimates of the prevalence and
incidence of four curable sexually transmitted
infections in 2012 based on systematic review
and global reporting. PLoS One 10:1–17
Isolation of Chlamydia-Specific B Cells
27