An improved method on isolation and serial passage of Chlamydia pneumoniae from human peripheral blood mononuclear cells.
BACKGROUND
Conventional method for Chlamydia pneumoniae (Cpn) isolation and propagation is technically challenging and time-consuming. Here, we developed a method to improve the isolation and passage of Cpn collected from human peripheral blood mononuclear cells (PBMCs).


METHODS
PBMCs positive with Cpn antigen (Cpn-Ag) were isolated, then centrifuged and cultured with Hep-2 cells after being broken. Cells were broken again and put into new Hep-2 cells to finish totally four passages with isolated and imported Cpn. Microimmunofluorescence method was used to detect Cpn. Inclusion forming unit (IFU) number was counted for each passage. Polymerase chain reaction (PCR) method was used to detect Cpn DNA. Efficiency of different centrifugation modes was compared.


RESULTS
Hep-2 cells of the first and second passages were strong positive with Cpn-Ag, the third passage was positive, and the fourth negative. Degeneration appeared in the fourth passage for isolated Cpn and third passage for imported strain. Centrifugation mode of 1,000 rpm for 2 h was the most efficient for Cpn propagation and passage.


CONCLUSIONS
This simplified method achieved efficient isolation, propagation, and passage of Cpn from PBMCs, and isolated strain was superior to imported strain on propagating ability.
Department of Respiratory Medicine, Tongde Hospital of Zhejiang Province, Hangzhou, Zhejiang, China.
Qian
Q
Jin
Feihua
F
Huang
Shuming
S
Sun
Ying
Y
Zhou
Xianrong
X
Xu
Weixing
W
Xi
D002460:Cell Line; D002470:Cell Survival; D002478:Cells, Cultured; D002498:Centrifugation; D016993:Chlamydophila pneumoniae / Q000302:isolation & purification*; D005455:Fluorescent Antibody Technique / Q000379:methods; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D012692:Serial Passage / Q000379:methods*; D014343:Trypan Blue
D014343:Trypan Blue
Chlamydia pneumoniae; centrifugation; immunofluorescence; inclusion bodies; polyethylene glycol