Streptavidin apc

The role of O-GalNAc-glycoproteins for rmE-selectin binding was investigated by adding 20 mM GalNAc-α-O-benzyl (Sigma) for 72 h to the standard tumor cell culture prior to the E-selectin binding assay. Likewise, tumor cell cultures were treated for 1 h with 10 mU/mL neuraminidase from Clostridium perfringens (Roche, Mannheim, Germany) or with 1 mg/mL pronase from Streptomyces griseus (Roche) under serum-free conditions at 37 °C for 1 h or 45 min before the E-selectin binding assay to analyze the role of sialic acid residues or glycoproteins, respectively. Detrimental effects of all treatments were excluded by propidium iodide staining immediately before flow cytometry. To test which linkage of sialic acid was mainly affected by neuraminidase, lectin binding flow cytometry was performed using biotinylated Maackia amurensis lectin II (MAA-II, detecting α-2,3-linked sialic acid) and Sambucus nigra agglutinin I (SNA-I, detecting α-2,6-linked sialic acid) (both from Vector Labs, Burlingame, USA). Both lectins were marked with streptavidin-APC (Sigma) before incubation with the tumor cells. To test for carbohydrate-specific binding, one tumor cell sample was pre-treated with periodic acid, which oxidizes free sialic acid residues and by this cracks the ring structure of the carbohydrate, which is required for the binding of MAA-II and SNA-I.

Altered membrane phospholipid asymmetry and RBC ageing was evaluated with Annexin-V (Life Technologies, Canada) and anti-CD47 (BioLegend, clone miap301) staining. Briefly, 1 μL of blood was collected in 1 mL of PBS, and centrifuged. Pellet was resuspended in Annexin binding buffer (Life Technologies), stained with FITC-labeled Annexin-V, PE-labeled rat anti-mouse CD47 monoclonal antibody and streptavidin-APC, and incubated at 20°C, in the dark for 15 min. Cells were washed and resuspended in PBS prior to FACS analysis of geometric mean of fluorescence intensity (gmeanFI) relative to Annexin-V and CD47 in streptavidin positive cells. Intra-erythrocytic ROS were measured using a modification of the Amer method [25 (link)]. Briefly, 2 x 10⁶ RBCs/mL were incubated at 37°C for 15 min in the dark with streptavidin-APC and 2’,7’-dichlorofluorescein diacetate (DCFDA; Sigma-Aldrich) dissolved in methanol. Cells were washed in PBS and analyzed for gmeanFI of 2’,7’-dichlorofluorescein (DCF) within streptavidin positive cells. Positive controls for Annexin-V and CD47 binding, and ROS measurement were achieved with a 15 min cells treatment with 50 μM hydrogen peroxide prior to washing and labeling.

The canonical E-selectin ligands were analyzed at the tumor cell surface by flow cytometry using mAbs against sLeX (CSLEX1) or sLeA (121SLE) as described.⁵¹ (link) β-1,6-GlcNAc branches and poly-LacNAc were determined using biotinylated PHA-L and DSL, respectively (Vector Labs). As “isotype” controls, lectins were applied after sugar inhibition with bovine thyroglobulin (Sigma) and chitin hydrolysate (Vector Labs), respectively. Lectins were labeled with streptavidin-APC (Sigma) for flow cytometry.
To test their functional importance for endothelial adhesion and E-selectin binding, sialic-acid-containing sugar residues were enzymatically cleaved using 10 mU/mL ND (from Clostridium perfringens, Roche) added to the tumor cell culture at 37°C for 1h under serum-free conditions. Likewise, glycoproteins were cleaved by 1 mg/mL pronase (from Streptomyces griseus) added to the tumor cell culture at 37°C for 45 min under serum-free conditions. O-GalNAc-glycosylation was chemically inhibited by adding 2 mM GOB (Sigma) for 72 h to the culture medium (solvent control: cell culture medium). N-glycosylation was inhibited using 2 μM synthetic Sw (Sigma) for 72 h (solvent control: methanol). Detrimental effects of all treatments on tumor cell viability were excluded by propidium iodide uptake analyses (flow cytometry [FC]).