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DAPI staining and flow cytometry analysis

LS Li Song
JP Junfeng Pan
YY Yantao Yang
ZZ Zhenxing Zhang
RC Rui Cui
SJ Shuangkai Jia
ZW Zhuo Wang
CY Changxing Yang
LX Lei Xu
TD Tao G. Dong
YW Yao Wang
XS Xihui Shen
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To perform DAPI staining and flow cytometry analysis49, overnight culture of E. coli BL21(DE3) containing the pET28a plasmid or its derivatives expressing Tce1 alone (pET28a-tce1) or Tce1-Tci1 together (pET28a-tce1-tci1) were diluted 100-fold into LB broth and incubated at 26 °C with 180 rpm shaking. After incubated at 26 °C for 2 h, the expression of toxin and immunity genes was induced by addition of 0.5 mM IPTG and continue cultivation for 4 h at 26 °C. Collected cells were washed with PBS, fixed, incubated for 5 min in PBS with 0.3% Triton X-100 stained using 10 μg ml−1 DAPI for 30 min at 37 °C (Solarbio, China), then washed three times with PBS and detected by fluorescence microscope (Andor Revolution-XD, Britain) or flow cytometry (Beckman, CytoFLEX). Twenty-thousand cells were gathered for each sample and analyzed by FlowJo_V10.

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