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Detection of Lysosomal Membrane Permeabilization
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Anne-Marie Ellegaard, Line Groth-Pedersen, Marja Jäättelä
Due to the reliance on the fluorescence of the Alexa Fluor label in this method, one has to consider if any other fluorescence arising from autofluorescence of the cells, treatment with a fluorescent small molecule or overexpression of a fluorescently tagged protein might interfere with the detection of the Alexa Fluor. Luckily, dextran coupled to many different Alexa Fluor labels is available, which makes it possible to solve this issue.
House dust mite-induced endoplasmic reticulum stress mediates MUC5AC hypersecretion via TBK1 in airway epithelium
Published in Experimental Lung Research, 2023
Jun Deng, Hongmei Tang, Yun Zhang, Xiefang Yuan, Ning Ma, Hang Hu, Xiaoyun Wang, Chunfeng Liu, Guofeng Xu, Yuejiao Li, Songping Wang, Linlin Guo, Xing Wang
BEAS-2B cells were treated as described above, and immunofluorescence staining was performed per standard laboratory protocol.23 Briefly, cell samples collected from chamber slides and sections of lung tissue were fixed with 4% formaldehyde and permeabilized with 0.3% Triton X-100 in PBS for 10 min at room temperature that around 20 °C. After blocking with 1% bovine serum albumin (BSA), the specimens were incubated with primary antibodies overnight at 4 °C. Alexa Fluor (555 or 488)-conjugated secondary antibodies were used to probe the primary antibody. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. Samples were then imaged and quantified using an SP5 Leica confocal microscope with Leica Application Suite Software (Version number: 14.0.0.162, Leica, Germany). The specific primary antibodies used in this study were MUC5AC (1:50; Abcam, Cambridge, UK, ab24070), NF-κB (1:200; Cell Signaling Technology, United States, 8242S), STAT6 (1:200; Cell Signaling Technology, United States, 5397S), BIP (1:200; Abcam, United Kingdom, ab21685), CHOP (1:100; Cell Signaling Technology, United States, 2895S), followed by staining with Alexa Fluor 555- or Alexa Fluor 488-conjugated secondary antibody (1:500; Invitrogen, United States, cat. no. A32727, A32732, and A28175). DAPI (Beyotime Institute of Biotechnology, China, C1002) was used to stain the nuclei. The mean fluorescence intensity was calculated using ImageJ software. Images were obtained from at least six random fields, and all images were used for qualification.
Elimination of plasma soluble antigen in cynomolgus monkeys by combining pH-dependent antigen binding and novel Fc engineering
Published in mAbs, 2022
Yuji Hori, Ken Ohmine, Hitoshi Katada, Yuki Noguchi, Kazuki Sato, Takeru Nambu, Lam Runyi Adeline, Gan Siok Wan, Kenta Haraya, Kazuhisa Ozeki, Masahiko Nanami, Tatsuhiko Tachibana, Zenjiro Sampei, Taichi Kuramochi, Junichi Nezu, Kunihiro Hattori, Tomoyuki Igawa
It is known that the clearance of antigen–antibody immune complexes from plasma is mainly mediated by liver sinusoidal endothelial cells and/or Kupffer cells that express FcγRIIb.38,39 To elucidate the underlying mechanism of the effect of the charge substitutions on the in vivo antigen sweeping, we evaluated the binding and the cellular uptake of the immune complex by Chinese hamster ovary (CHO) cells that did or did not stably express huFcγRIIb. Alexa Fluor 488-labeled antigen and the tested antibodies, pH-IgG1, pH-V3, pH-pI(A), and pH-V3-pI(A), were incubated with the cells, and the cellular fluorescence intensity was measured by flow cytometer. Compared with pH-IgG1, pH-V3 and pH-pI(A) showed significantly higher binding and uptake, and that pH-V3-pI(A) was especially high. In contrast, they show little binding and uptake in the parent CHO cells (Figure 5). Furthermore, the enhanced binding and uptake by the Fc-engineered variants, not only in pH-V3, but also in pH-pI(A) and pH-V3-pI(A), were completely inhibited by an anti-huFcγRIIb antibody (2B6) that recognizes the Fc binding site of huFcγRIIb,40 although they were not inhibited by the isotype control antibody (Figure 5). These results indicate that the positive-charge substitutions further enhance FcγRIIb binding and increase FcγRIIb-mediated cellular uptake, rather than nonspecific uptake.
An affinity threshold for maximum efficacy in anti-PD-1 immunotherapy
Published in mAbs, 2022
Sarah C. Cowles, Allison Sheen, Luciano Santollani, Emi A. Lutz, Brianna M. Lax, Joseph R. Palmeri, Gordon J. Freeman, K. Dane Wittrup
Samples were prepared with 10 μL of labeled beads in 0.3–0.5 mL of running buffer (PBS (Corning) with 0.01% v/v Tween-20 (Millipore Sigma)) with at least a 10 nM concentration of Alexa Fluor 488-labeled murine PD-1 monoFc fusion protein. Samples were incubated with continuous rocking at room temperature for 20–60 minutes. The beads were then washed three times and resuspended with a concentration of 10x excess unlabeled murine PD-1 monoFc fusion protein in 1 mL of running buffer. The samples were then incubated at 37°C until the various time points were reached. At each time point, a small volume of sample was removed (~50 μL) and washed three times. The sample was then resuspended in PBS (Corning) with 1 mg/mL BSA (Thermo Scientific Fisher) for flow cytometry. Controls were prepared to calculate the background signal (no Alexa Fluor 488-labeled antigen exposure) and 100% signal (time zero sample). Median fluorescence intensity in the Alexa Fluor 488 channel was determined using FlowJo_v10.7.2 (FlowJo, LLC). MATLAB (R2019b, Mathworks) was used to plot and derive an apparent koff rate using a single exponential decay equation.