5x alphalisa lysis buffer  (Revvity)

Journal: Pharmaceutics

Article Title: Optimization and Characterization of Novel ALCAM-Targeting Antibody Fragments for Transepithelial Delivery

doi: 10.3390/pharmaceutics15071841

Figure Lengend Snippet: Smaller antibody formats more effectively penetrate the human corneal epithelium and remain functional following penetration. ( A ) Schematic depiction of the 3D human epithelial corneal tissue model grown in cell culture inserts. IF8-Fc, db V2D7, and scFv V2D7 were applied topically on the corneal tissue layer at a concentration of 500 nM and left to penetrate for 6 h. ( B ) H&E-stained cross-section of 3D human corneal epithelial tissue model (upper image) and human corneal tissue (bottom image). AL: apical layer; WC: wing cells; BL: basal layer; M: microporous membrane; S: stroma ( C ) Percentage (%) of the antibody compared to the initial input or ( D ) antibody concentration (nM) that crossed the corneal epithelial layer after 6 h determined with an AlphaLISA assay. Representative plots of one out of two experiments performed. Mean of n = 3–8 replicates. ( E ) Human corneal epithelial cells express ALCAM, as assessed by flow cytometry (n = 1). ( F ) Transepithelial electrical resistance (TEER) of the 3D corneal tissue model measured after the penetration assay. Representative plot of one out of two experiments performed. Mean of n = 4 replicates. ( G ) Schematic depiction of the dendritic cell (DC) transmigration assay performed in transwells containing a lymphatic endothelial cell (LEC) monolayer. The medium from the penetration assay containing mAbs that crossed the corneal epithelial layer was applied to the top of the transwell to assess its ability to block DC transmigration towards the chemokine CCL21. ( H ) DC transmigration performed in the presence of the medium collected from the corneal tissue penetration assays (see A – D ) with scFv, db and IF8-Fc (one technical replicate out of two shown) or negative control PBS (n = four transwells per condition). IF8-Fc at 1 nM was used as a positive control, and PBS was used as the negative control (control). Representative plot of one out of two experiments. Statistics: One-way ANOVA, Holm–Sidak multiple comparison test ( C , D ) or Dunnett’s multiple comparison test ( H ).

Article Snippet: In brief, 10 µL of 10 nM hALCAM V1 diluted in 1X AlphaLISA buffer (AL000C, PerkinElmer) was added to wells of a 96-well plate (6002350, PerkinElmer).

Techniques: Functional Assay, Cell Culture, Concentration Assay, Staining, Membrane, Flow Cytometry, Transmigration Assay, Blocking Assay, Negative Control, Positive Control, Control, Comparison