Cell separation was performed using a BD Facs Aria III cell sorter with cells sorted directly into chilled cell lysis and RNA stabilisation buffer (Buffer RLT, Qiagen) prior to storage at −80° C. Sort purity was verified to >99% prior to and after each sort.
Beads were washed three times with lysis buffer prior to use.
Positive ccs were buffer-exchanged into carbonate buffer prior to purification using a stirred ultrafiltration cell (Amicon) or by dialysis.
After binding, beads were washed three times for 5 10 minutes with wash buffer prior to addition of sample buffer.
Beads were washed twice with HEPES buffer and once with lyses buffer prior to incubation with cell extracts.
Beads were washed 3 X in PB buffer and resuspended in 2X Laemelli sample buffer prior to western blotting.
RBCs were lysed with ammonium chloride buffer prior to staining.
Protease inhibitors were added to all buffers excluding the urea buffer prior to use.
Deglycosylated ovalbumin was buffer exchanged into aqueous AmAc buffer prior to CIP dephosphorylation.
Cell debris was removed by centrifugation at 250 g for 5 min and the supernatant containing partially purified chlamydiae was mixed with an equal volume of phosphate buffer containing 0.4 M sucrose (2SP), prior to storage at -80°C.
