H1N1-virus-specific antibody-secreting cells (ASCs) in the spleens of H1N1-challenged WT and db/db mice were identified by ELISPOT64 (link). Briefly, 96-well plates (Cat. # MSIPS4W10, Millipore) were pre-coated with purified H1N1 influenza virus A/PR/8/34 (5 mg/mL) at 4 °C overnight. Plates were washed and blocked with RPMI 1640 with 10% FBS at room temperature for 1 h. 1 × 104 PI-CD3-B220+ cells from spleens of WT and db/db mice with H1N1 influenza virus on 9 days post-challenge were sorted and cultured for 12 h. Goat Anti-Mouse IgG Alkaline Phosphatase was then added after thoroughly wash and incubated for 1 h at room temperature. 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP)/Nitro Blue Tetrazolium (NBT) substrate solution (Sigma–Aldrich) was added. Spots were monitored, stopped by gently washing, identified and counted by the Photoshop CS4 software (Adobe).
Deng J., Chen Q., Chen Z., Liang K., Gao X., Wang X., Makota F.V., Ong H.S., Wan Y., Luo K., Gong D., Yu X., Camuglia S., Zeng Q., Zhou T., Xue F., He J., Wei Y., Xiao F., Ma J., Hill D.L., Pierson W., Nguyen T.H., Zhou H., Wang Y., Shen W., Sun L., Li Z., Xia Q., Qian K., Ye L., Rockman S., Linterman M.A., Kedzierska K., Shen N., Lu L, & Yu D. (2021). The metabolic hormone leptin promotes the function of TFH cells and supports vaccine responses. Nature Communications, 12, 3073.
5 bromo 4 chloro 3 indolyl phosphateAlkaline phosphatase Anti igg Antibody secreting cellsCellsGoat H1n1 influenza virus Influenza a h1n1 virus Mouse Nitro blue tetrazolium Spots
Corresponding Organization :
Other organizations :
Shanghai Jiao Tong University, Renji Hospital, City University of Hong Kong, Shenzhen Research Institute, University of Hong Kong, Australian National University, Huashan Hospital, Fudan University, Peking University People's Hospital, Peking University, Shandong Academy of Sciences, Qilu University of Technology, Babraham Institute, University of Melbourne, Peter Doherty Institute, Nanjing Drum Tower Hospital, Army Medical University
Number of H1N1-virus-specific antibody-secreting cells (ASCs) in the spleens of H1N1-challenged mice
control variables
Time of sample collection (9 days post-challenge)
Cell type (PI-CD3-B220+ cells)
Cell culture duration (12 h)
Purified H1N1 influenza virus A/PR/8/34 concentration (5 mg/mL) used for plate coating
RPMI 1640 with 10% FBS used for blocking
Goat Anti-Mouse IgG Alkaline Phosphatase used for detection
BCIP/NBT substrate solution used for visualization
positive controls
Not explicitly mentioned
negative controls
Not explicitly mentioned
Annotations
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