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. Author manuscript; available in PMC: 2011 Dec 15.
Published in final edited form as: J Immunol Methods. 2010 Oct 1;363(1):9–20. doi: 10.1016/j.jim.2010.09.025

Figure 2. Expression of collagen by 5 day cultured spleen cells.

Figure 2

ACK-treated spleen cells were cultured for 5 days at 3.5 × 105 cells per well in the presence of IL-13 and M-CSF. Adherent cells were removed by trypsin-EDTA treatment and stained for the presence of CD11b. The cells were then fixed, permeabilized, stained with rabbit polyclonal antibodies, and analyzed by flow cytometry. A) Forward and side scatter characteristics of 5 day cultured spleen cells. (B) Forward and side scatter analysis of CD11b+ cells. C and D) Histograms show fluorescence intensity of FITC-conjugated goat F(ab′)2 anti-rabbit 2° (black line) compared to collagen I (red line), collagen III (blue line), and as a positive control syk (yellow line). Flow cytometry plots are representative of 3 independent experiments. C) When gating the entire live cell population R1 (from A), collagen was nearly undetectable, but when gating (D) for CD11b+ cells and region P1 (from A and B) there was an increase in the levels of both collagen I and III. E) Compared to FITC control, CD11b+ cells had a significant increase in median fluorescent intensity for collagen I and III staining (1-way ANOVA, Dunnett’s test). **, p < 0.01; ***, p < 0.001.