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Blood, Vol. 111, Issue 2, 894-904, January 15, 2008
Extravasations and emigration of neutrophils to the inflammatory site depend on the interaction of immune-complex with Fc receptors and can be effectively blocked by decoy Fc receptors
Blood Shashidharamurthy et al.
111: 894
Supplemental materials for: Shashidharamurthy et al
Files in this Data Supplement:
- Figure S1 (PDF, 15.7 KB) -
(A) P388D1 (50 µl of 5 × 106) cells were incubated in the presence and absence of mIgG2a (50 µg/ml). The binding of mIgG2a to the P388D1 cells were confirmed by staining the cells with FITC conjugated F(ab′)2 of goat anti mouse-IgG. (B) Soluble IC binding assay was carried out as described in the text. Briefly, the macrophages (50 µl of 5 × 106) were incubated with FITC-IC (20 µg/ml) in binding buffer (pH 7.4) for 1 h at 4°C in the presence or absence of purified blocking mAbs. Cells were preincubated F(ab′)2 and intact antibody of 2.4G2 for 30 min at 4°C. The F(ab′)2 mAb was used to block sIC binding mediated by CD16A/32B and intact mAb to block total binding. Monomeric mIgG2a was used to block CD64. The cells were then washed and resuspended in 150 µl of binding buffer and fixed by adding 150 µl of 2% formalin in PBS. Binding of FITC-IC to macrophages were analyzed by flow cytometry.
- Figure. S2. CD16A-Ig blocks the EA binding and phagocytosis by mouse peritoneal macrophage cells (PDF, 21.1 KB) -
(A) CD16A-Ig blocked the binding of particulate IC to the mouse Thioglycolate elicited peritoneal macrophage cells in a dose dependent manner. Peritoneal macrophage cells were incubated with PKH-labeled-EA (particulate IC) in the presence of various concentration of CD16A-Ig. EA bound to the cells were analyzed by flow cytometry. Cells incubated with PKH-labeled unopsonized-E were used as a background control. The EA binding in medium was taken as 100% to calculate the percent EA binding. Data shown are the average (mean ± SD) of three individual experiments. (B) Thioglycolate elicited peritoneal macrophage cells (1 × 105 in 50 µl) were incubated with PKH-labeled-EA (50 µl of 1.5 × 108 cells) for 2 h at 4°C. EA binding was performed in the presence of the following blocking reagents: mIgG2a (50 µg/ml), anti-CD16A/CD32B mAb (2.4G2; 10 µg/ml), CD16A-Ig (25 µg/ml), CD32A-Ig (25 µg/ml), or a combination of CD16A-Ig and CD32A-Ig (25 µg/ml each). EA bound to the cells were analyzed by flow cytometry. Values are Mean ± SD of data from three individual experiments (*P<0.01). (C) Phagocytosis of Alexa-EA was carried out using mouse peritoneal macrophage cells. 50 µl of 5 × 106 cells/ml of macrophages were incubated in a tube with 50 µl of 1.5 × 108 cells/ml of Alexa-EA in the absence or presence of the blocking reagents 2.4G2 (10 µg/ml) and CD16A-Ig (25, 50 and 75 µg/ml) on ice for 1 h. Cell surface-bound Alexa-EA were lysed by a 25 sec hypotonic shock, and the cells were then washed twice with binding buffer and fixed with 1% formalin for 10 min at 4°C. To quench surface fluorescence, 0.2% methylene blue was added to the samples. Phagocytosed Alexa-EA were counted using flow cytometry. Cells incubated with EA at 4°C served as negative control. The phagocytic index (PI) was calculated as described in the text. The experiment was repeated twice (*P<0.01) and the data are average of two experiments.
- Figure S3. Effect of cobra venom factor on hemolytic activity mediated by mouse complement (PDF, 13.5 KB) -
The hypocomplementemia was induced in mice by injecting i.p with two doses of 150 U cobra venom factor (CVF) at 8 h intervals. The depletion of complement system was confirmed by measuring the hemolytic activity using mouse serum. Briefly, rabbit red blood cells (rRBC) were coated with DNP as described in the text for SRBCs and opsonized by incubating with rabbit anti-DNP-IgG for 1 h at room temperature, washed three times with DGV buffer and stored in the same buffer. The rabbit anti-DNP IgG opsonized rRBCs were incubated with various dilutions of mouse serum from both normal and CVF treated mice for 1h at 37°C. The reaction was stopped by adding 0.8 ml of ice-cold DGV buffer. Cells with buffer alone served as controls. Cells lysed with H2O served as 100% lysis. All the samples were centrifuged for 5 min at 2000 rpm at 4°C. The supernatant was collected and read at 450nm. As shown in the figure, about 80 % of the activity was abolished in CVF treated mice, suggesting the depletion of complement activity by CVF.
- Figure S4. Titration of mouse serum for complement activity (PDF, 12.2 KB) -
Complement-mediated hemolytic activity was carried out as described earlier (Fig. S3). The opsonized rRBCs (100µl of 2 × 108 cells/ml) were incubated with 100 µl of various dilutions of mouse serum for 1hr at 37°C. The reaction was stopped by the addition of 0.8 ml of ice-cold DGV buffer. Cells with buffer alone served as controls. All the samples were centrifuged for 5 min at 2000 rpm at 4°C. The supernatant was collected and read at 450nm. As shown in the figure, up to 1:20 dilution there is measurable hemolytic activity and at 1:5 dilution 50 % of the rRBCs lysed and therefore we have used this dilution for all the experiments.
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