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Cell Biology International (2012) 36, 223–228 (Printed in Great Britain)

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Perforin is recaptured by natural killer cells following target cells stimulation for cytotoxicity

Lei Wang*†, Rulin Sun*, Pan Li*, Yang Han*, Ping Xiong*, Yong Xu*, Min Fang*, Zheng Tan*, Fang Zheng*¹ and Feili Gong*¹

*Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Peoples Republic of China, and †Institute of Laboratory Medicine, Hubei University of Chinese Medicine, No. 1, Huangjia Lake West Road, Wuhan, 430065, Peoples Republic of China

¹Correspondence may be addressed to either of the authors (email zhengfangtj@yahoo.com or flgong@163.com).

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SUPPLEMENTARY ONLINE DATA

Figure S1 CPZ treatment does not affect the degranulation of NK92 cells

NK92 cells were pretreated with CPZ for 30 min and were kept in the medium for 2 h. K562 (4×10⁵) and NK92 (2×10⁵) cells were mixed in a volume of 200 μl and centrifuged at 100 g for 1 min to enhance cell contact. After co-incubation for 2 h, the cells were harvested and stained by FITC-conjugated anti-CD107a Ab and APC-labelled anti-CD56 Ab. The CD107a⁺/CD56⁺ double-positive cells indicated the degranulated NK92 cells. CD107a⁺ percentage shows the degranulation rate of NK92 cells. MFI means the mean fluorescence intensity of CD107a.

Figure S2 Pretreatment with CPZ attenuates the endocytosis of transferrin in NK92 cells for 4 h

NK92 cells were pretreated with CPZ for 30 min, centrifuged to discard CPZ in the supernatant and incubated in 100 μl of ice-cold α-MEM containing 10 μg/ml Alexa Fluor-488-transferrin (Molecular Probes, Invitrogen) at 4°C for 30 min. Cells were then centrifuged at 400 g for 5 min to discard the supernatant. Pre-warmed 10% FCS-α-MEM was then added to resuspend the cell pellet and incubated at 37°C for 4 h to allow the internalization of membrane-bound transferrin. After externally bound transferrin was removed with ice-cold acid wash buffer (150 mM NaCl and 25 mM acetic acid, pH 3) at 4°C for 3 min, the cells were washed and measured by flow cytometry. Control: after centrifugation at 400 g for 5 min to discard supernatant, Alexa Fluor®-488–transferrin-labelled NK92 cells were kept in ice-cold α-MEM at 4°C continuously and without being washed with ice-cold acid wash buffer. MFI means the mean fluorescence intensity of internalized transferrin. **P<0.01.

Figure S3 Clathrin in NK92 cells was reduced by specific siRNA tranfection

CHC, cells transfected with siRNA targeting clathrin heavy chain; control, non-transfected cells; scrambled, cells transfected with scrambled siRNA.

Figure S4 Treatment with CPZ does not affect the viability of NK92 and target cells

NK92 cells (A), K562 cells (B) and PECs (C) were treated with or without CPZ for the indicated times.

Received 2 May 2011/11 September 2011; accepted 10 October 2011

Published as Cell Biology International Immediate Publication 10 October 2011, doi:10.1042/CBI20110242

© The Author(s) Journal compilation © 2012 Portland Press Limited