For assessment of adhesion, the human neutrophil suspension (1x107 cells/ml) was loaded with Calcein-AM to a final concentration of 1 μM prior to incubation for 45 min at 37 °C. The labeled neutrophils (1x106 cells/well) were incubated with sAGP-1 or nAGP-1 (25, 50 and 100 µg/ml) in triplicate wells in cell culture plates (Nest Biotechnology Co. Ltd., China) pre-coated with 0.2% gelatin. Unbound neutrophils were removed by washing twice with HBSS/A and the adherent neutrophils were visualized and photographed at a magnification of 10x by fluorescent microscopy (Motic BA410 fluorescence microscope, Hong Kong; with Nikon DS-Qi2 camera, Japan) [36 (link)]. The number of cells adhered in each well was determined by counting the cells adhered in 10 randomly chosen fields using ImageJ software (ver.1.51j8) and then calculating the average number of cells adhered per field.
Protocol detail
Quantifying Neutrophil Adhesion with AGP
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Corresponding Organization : University of Mysore
Other organizations : University of Utah, University of Michigan–Ann Arbor, Hadassah Medical Center, Beth Israel Deaconess Medical Center, Harvard University, Cleveland Clinic Lerner College of Medicine, Geriatric Research Education and Clinical Center, George E. Wahlen Department of VA Medical Center
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Variable analysis
independent variables
- SAGP-1 concentration (25, 50 and 100 µg/ml)
- NAGP-1 concentration (25, 50 and 100 µg/ml)
- Number of adherent neutrophils per field
- Human neutrophil suspension concentration (1x10^7 cells/ml)
- Calcein-AM concentration (1 μM)
- Incubation time (45 min)
- Incubation temperature (37 °C)
- Neutrophil cell density (1x10^6 cells/well)
- Gelatin coating concentration (0.2%)
- Washing buffer (HBSS/A)
- Microscope magnification (10x)
- Image analysis software (ImageJ)
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