E selectin fc chimera

Design and assembly of the microfluidic device and the whole blood adhesion assay were performed as previously reported.^{24 (link)} Briefly, clean coverslips that were coated with recombinant mouse VCAM-1/Fc chimera and E-selectin/Fc chimera (R&D Systems) were assembled with a 4-channel PDMS device and kept on a 37°C surface. Peripheral blood (0.1 mL) was drawn into heparin-coated tubes via facial vein puncture and incubated with PE–anti-CD115 and FITC–anti-CD11c antibodies (with corresponding isotypes as negative controls, Supplemental Figure II) at room temperature for 20 min. Then, after 1:3 dilution in PBS with calcium and magnesium, 60 μL diluted blood was introduced into the channel at a flow rate that produced a shear stress of 2 dynes/cm² at fluid–glass interface. Blood was perfused through the chamber for 5 min followed by fixation and mounting in medium containing DAPI. The number of adherent “foamy” (CD115⁺CD11c⁺) monocytes was counted and normalized by total infused monocyte number.

Venous blood into EDTA or ACD drawn immediately pre- and 24±4 hours post- infusion was evaluated by flow cytometry for adhesion markers on CD16b+ (ID3, Beckman Coulter) neutrophils: activated Mac-1 (CBRM1/5, eBioscience) or activated β2 integrin (ab13219, abcam), Mac-1 (ICRF44, BD Pharmingen), CD44 (G44-26, BD Pharmingen), E-selectin-Fc chimera (R&D Systems), L-selectin (DREG-56, eBioscience), LFA-1 (HI111 BD Pharmingen), and PSGL-1 (KPL-1, BD Pharmingen). Activated Mac-1 Ab detects the functionally active form of Mac-1(was performed on 200–800 mg/kg cohorts) and activated β2 integrin recognizes the high affinity conformation of β2 integrin, the β subunit of LFA-1 and Mac-1 (was performed on 100 mg/kg cohort).
With late afternoon or evening study drug administration, samples were stored overnight as needed (range 8–24 hours) at 4°C prior to analysis. When stored overnight, all paired pre-IVIG and 24 hour post-IVIG samples per patient were stored for uniform lengths of time. No adhesion markers except activated Mac-1 show expression increase at 24 hr compared to immediate analysis (Figure 1B). Since the time interval to analysis was constant for each subject’s two samples, the ratio between the two samples (Figure 1A) was unaffected by storage.

Non-tissue culture treated polystyrene petri dishes (Corning, Corning, NY) were enclosed using a single well FlexiPerm gasket (Sigma-Aldrich, St. Loius, MO). A solution of 2 μg mL⁻¹ of Protein A/G (Themo-Fisher Scientific, Waltham, MA) and 1 μg mL⁻¹ of SDF-1α (R&D Systems, Minneapolis, MN) was applied and incubated overnight at 4°C. The surfaces were then washed three times with PBS before a 30 minute incubation of a 0.2% w/v solution of Pluronic F-127 (Sigma-Aldrich). The surfaces were washed again three times with PBS. Next, a solution of ICAM-1/Fc and/or E-selectin/Fc chimera (R&D Systems) was applied to the surface and incubated for three hours at room temperature. The completed surfaces were then washed again three times with PBS before use. For experimentally tested points, molar ratios of 1:0, 10:1, 1:1, 1:10, and 0:1 E-selectin/Fc:ICAM-1/Fc were chosen to highlight differences between similar surfaces. In addition, total protein concentrations were chosen to yield total site densities of 4–5, 35–45, 130–160, 310–360, 550–610, and 1200–1300 sites/μm².