igg2 isotype control  (Bio X Cell)

Journal: bioRxiv

Article Title: Adenovirus phagocytosis by neutrophils triggers a pro-inflammatory response

doi: 10.1101/2025.09.02.673675

Figure Lengend Snippet: (A, B) Alexa-488 (A-488) labeled Ad5 or A-488 Ad3 were incubated at 37°C with or without human serum (HS), heat-inactivated (HI-HS) or IgG-depleted HS. Then PMNs were exposed to Ads at 4°C to allow cell binding (MOI 10 4 vp/cell) and analysed by flow cytometry. The results are presented as an association index ± SEM ( i.e . mean fluorescence multiplied by percentage of positive cells) (n ≥ 3). (A) Ad association index without serum. (B) Ad association index in the different conditions described above. Mann-Whitney tests: *, p < 0.05; **, p < 0.01. (C, D) A PMN model cell line, PLB-985 cells, was untreated or incubated with blocking antibodies against CD16 and/or CD32 receptors, or with control isotypes. Then cells were incubated with HS-pre-incubated A-488 Ad5 (C) or Ad3 (D) at 4°C to allow binding. Results show the percentage of binding inhibition (± SEM) for the different Ad + HS conditions (five independent experiments) relative to untreated cells exposed to HS-opsonized Ad: Mann-Whitney test * p < 0.05; ** p < 0.01.

Article Snippet: For Fcγ receptor-blocking experiments, the same protocol was used except that cells were also pre-incubated for 20 min at 4°C with the following blocking antibodies : anti-CD16 [3G8] clone antibody (ARG42244, ArigoBio) 5 μg/mL and/or anti-CD32 IV.3 clone antibody (BioXCell BE0224) 10 μg/mL, or control isotypes IgG1 (ARG20767, ArigoBio) 5μg/mL and IgG2 (MPC-11 clone, BioXCell BE0086) 10 μg/mL.

Techniques: Labeling, Incubation, Binding Assay, Flow Cytometry, Fluorescence, MANN-WHITNEY, Blocking Assay, Control, Inhibition

Journal: bioRxiv

Article Title: Adenovirus phagocytosis by neutrophils triggers a pro-inflammatory response

doi: 10.1101/2025.09.02.673675

Figure Lengend Snippet: IgG-coated Ads bind to PMNs via CD32 receptor recognition. This first step triggers Ad internalization in a CD63 + Rab5 + phagosome. Simultaneously, an Ad-specific transcriptional program is induced while NOX2 produces ROS, and an extracellular calcium entry occurs via calcium (or cationic) channels. These events lead, after a few hours, to the PMN lytic death with NET emission, and IL-8 release. This cell death depends on extracellular calcium and RIPK3 (created with BioRender).

Article Snippet: For Fcγ receptor-blocking experiments, the same protocol was used except that cells were also pre-incubated for 20 min at 4°C with the following blocking antibodies : anti-CD16 [3G8] clone antibody (ARG42244, ArigoBio) 5 μg/mL and/or anti-CD32 IV.3 clone antibody (BioXCell BE0224) 10 μg/mL, or control isotypes IgG1 (ARG20767, ArigoBio) 5μg/mL and IgG2 (MPC-11 clone, BioXCell BE0086) 10 μg/mL.

Techniques: