Journal: Nature Communications
Article Title: Programmed cell removal by calreticulin in tissue homeostasis and cancer
Figure Lengend Snippet: Identification of CRT-binding ligands on aged and malignant cells. a Screening for CRT-binding glycans with carbohydrate microarray. Purified CRT-IgG-Fc proteins were used to probe a carbohydrate microarray containing a number of different types of glycans, including N-, O- and sulfated-glycans. Anti-IgG-Fc antibody was used for detecting binding of CRT to glycans. Glyco-antigens were used at 0.05 and 0.25 μg μl −1 (left and right bars for each glycan). n = 3. Error bars represent standard deviation. b PHA-L binding to peritoneal neutrophils and macrophages at 0, 4, 6, 8, 24, and 72 h after thioglycollate injection in MRP8-Bcl2 mice. c , d Examination of cell surface CRT-binding sites on neutrophils. Bone marrow ( c ) or peritoneal neutrophils ( d ) were collected and treated with heat inactivated neuraminidase (Δneu) or neuraminidase (neu). Cells were incubated with PBS (control; black) or recombinant CRT proteins (red) and binding of CRT was then measured by flow cytometry with PE-conjugated anti-CRT antibody. rCRT binds to mature peritoneal neutrophils but not the immature bone marrow neutrophils. Treatment with neuraminidase led to the release of CRT-binding sites. e , f Immunofluorescent staining of CRT in HL60 ( e ) and SW620 ( f ) cells. CRT localized to perinuclear regions, vesicles, and cell surface. CRT was either expressed at a low level ( e ) or limited to the perinuclear regions ( f ), while a significant portion of PHA-L staining were observed on the cell surface ( e and f ).In a – d , MFI, mean fluorescence intensity
Article Snippet: For CRT-binding experiments, cells were incubated with recombinant calreticulin human Fc fusion protein at 40 µg ml−1 or biotin-PHA-L 6 µg ml− 1 (B-1115, Vector laboratory) for 1 h incubated on ice.
Techniques: Binding Assay, Microarray, Purification, Standard Deviation, Injection, Mouse Assay, Incubation, Recombinant, Flow Cytometry, Cytometry, Staining, Fluorescence