Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

ICC/IF image of ab6046 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6046, 1µg/ml) overnight at +4°C. The secondary antibody (red) was ab150083 Alexa Fluor® 647 goat anti-rabbit IgG (H+L) used at 2µg/ml for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• Flow Cyt

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of HEK-293 (human embryonic kidney epithelial cell, Left) / A431 (human epidermoid carcinoma epithelial cell, Right) cells labelling HLA E with ab324677 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Low expression : HEK-293.
Cells were stained on ice to avoid internalization.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HL-60 (human acute promyelocytic leukemia promyeloblast, Left) / K-562 (human chronic myelogenous leukemia lymphoblast, Right) cells labelling CISH/CIS with ab324909 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Negative control : HL-60 (PMID : 11032736).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype (Left) / HEK-293T (human embryonic kidney epithelial cell) transfected with a Virion membrane protein A21 expression vector containing a GFP-tag (middle) / HEK-293T cells transfected with a empty expression vector containing a GFP-tag (Right) cells labelling Vaccinia Virus with ab312854 at 1/500 dilution (0.1 ug)/ Middle and Right (Red) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of K-562 (human chronic myelogenous leukemia lymphoblast, Left) / Jurkat (human T cell leukemia T lymphocyte from peripheral blood, Right) cells labelling HLA E with ab324677 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Low expression : K-562.
Cells were stained on ice to avoid internalization.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Human PBMC (human peripheral blood mononuclear cell) cells labelling Granzyme A with ab321992 at 1/50 dilution (1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Cells were co-stained with anti human CD56 conjugated to PE.

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of K-562 (human chronic myelogenous leukemia lymphoblast, Left) / Jurkat (human T cell leukemia T lymphocyte from peripheral blood, Right) cells labelling CD1d with ab324952 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Negative control : K-562 (PMID : 32324315).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Intracellular Flow Cytometry analysis of HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells transfected with a SARS-COV2 orf7a expression vector containing a GFP tag labeling SARS-CoV2 orf8 with ab283914 at 1/500 dilution (right). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti-rabbit IgG (Alexa Fluor® 647, ab150083) secondary antibody was used at 1/5000 dilution. Isotype control - Rabbit monoclonal IgG (left) (ab172730). No cross-reactivity with SARS-COV2 orf7a.

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype (Left) / 293T cells transfected with a Cholera enterotoxin subunit B expression vector containing an EGFP tag (Middle) / 293T cells transfected with an empty expression vector containing an EGFP tag (Right) cells labelling Cholera enterotoxin subunit B with ab317738 at 1/5000 dilution (0.01 ug)/Middle and Right (Red) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype (Left) / 293T cells transfected with a human HA Tag expression vector containing a GFP tag (Middle) / 293T cells transfected with an empty expression vector containing a GFP tag (Right) cells labelling HA tag with ab314237 at 1/5000 dilution (0.01 ug)/Middle and Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control.

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype (Left) / 293T cells transfected with a EGFP expression vector containing a EGFP tag (Right) cells labelling GFP with ab314656 at 1/500 dilution (0.1 ug)/Right (Red) compared with a Rabbit monoclonal isotype control - Alexa Fluor® 647 / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of Human PBMC (human peripheral blood mononuclear cell) cells labelling HLA E with ab324677 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Cells were stained on ice to avoid internalization.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) (Right) / 293T (human embryonic kidney epithelial cell) (Left) cells labelling Granzyme A with ab321992 at 1/500 dilution (0.01ug) / Right compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Negative control : 293T.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Human PBMC (human peripheral blood mononuclear cell) cells labelling Granzyme A with ab321992 at 1/50 dilution (1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Cells were co-stained with anti human CD8a conjugated to Pacific blue.

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of Human PBMC (human peripheral blood mononuclear cell) cells labelling CD1d with ab324952 at 1/500 dilution (0.1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Cells were co-stained with anti-CD3 conjugated to Brilliant Violet 421.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Intracellular Flow Cytometry analysis of HEK-293T (Human embryonic kidney epithelial cell) cells transfected with a SARS-CoV-2 nsp9 (Left) or SARS-CoV2 nsp16 (Right) expression vector containing a myc tag labeling SARS-CoV-2 nsp16 with ab284038 at 1/500 dilution (Left and Right). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 647, ab150083) secondary antibody was used at 1/5000 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype (Upper Left) / 293T (human embryonic kidney epithelial cell) transfected with an Granzyme A expression vector containing a myc-His-tag® (Upper Middle) / 293T cells transfected with an empty expression vector containing a myc-His-tag® (Upper Right) / 293T cells transfected with homologous human Granzyme expression vector (GrzmB, GrzmH, GrzmK, GrzmM) containing a myc-His-tag® (Lower). cells labelling Granzyme A with ab321992 at 1/50 dilution (1ug) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Cells were first stained with rabbit IgG or ab321992. After fixation and permeability, cells stained with anti-myc tag conjugated to Alexa Fluor® 647.
Crossreactivity with protein Granzyme B, Granzyme H, Granzyme K, Granzyme M were fully tested.

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometry overlay histogram of Negative control : HeLa (human cervical adenocarcinoma epithelial cells, Left) and IMR-32 (human neuroblastoma neuroblasts, Right) labelling GPC2 with ab324378 at 1/50 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Secondary antibody details, Goat anti-Rabbit IgG (Alexa Fluor^® 647 (ab150083), at 1/5000 dilution was used as the secondary antibody. Gated on viable cells.

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized Isotype (Row1) / 293T (human embryonic kidney epithelial cell) transfected with a human HLA-E expression vector containing a myc-His-tag® (Row1) / 293T transfected with a human HLA expression vector (HLA-A, HLA-B, HLA-C, HLA-G, HLA-H) or an empty expression vector containing a myc-His-tag® (Row2-4) cells labelling HLA E with ab324677 at 1/500 dilution (0.1ug) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control.

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Cells were surface stained with isotype control or our antibody. Then fixed with 2% PFA for 10min followed by intracellularly stained with anti-myc tag conjugated to Alexa Fluor® 594.
Cells were stained on ice to avoid internalization.

• Flow Cyt

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of MOLT-4 (human lymphoblastic leukemia T lymphoblast, Left) THP-1 (human monocytic leukemia monocyte, Right) cells labelling IL-17RA Receptor with ab325606 at 1/500 dilution (0.1ug) / Red compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells. Low expression : MOLT-4.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (human embryonic kidney epithelial cell) cells labelling MERS Spike glycoprotein with ab322146 at 1/500 dilution, followed by Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody (Alexa Fluor® 488) antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in 293T cells transfected with a MERS-CoV S protein expression vector containing a myc-His-tag®(shown in green) and no staining in 293T cells transfected with a SARS-CoV-2 Spike glycoprotein expression vector containing a myc-His-tag®. The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab9106 Anti-Myc Rabbit polyclonal antibody was used to counterstain tubulin at 1/200 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody (Alexa Fluor® 488) at 1/1000 dilution.

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of 786-O (human kidney epithelial cell, Left) DLD-1 (human colorectal adenocarcinoma epithelial cell, Right) cells labelling LOX 1 with ab325339 at 1/500 dilution (0.1ug) / Red compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody)/ Blue.

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells. Negative control : 786-O.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of HEK-293T (human embryonic kidney) cells transfected with human SIGLEC14 (Left) or SIGLEC5 (Right) expression vector containing GFP tag cells labeling SIGLEC5 and SIGLEC14 with ab307846 at 1/500 dilution (0.1µg). Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody. Cross-react with SIGLEC14. Gated on viable cells.

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometry overlay histogram of WERI-Rb-1 (human Eye Retina grape-like clusters of round cells) labelling GPC2 with ab324378 at 1/50 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Secondary antibody details, Goat anti-Rabbit IgG (Alexa Fluor^® 647 (ab150083), at 1/5000 dilution was used as the secondary antibody. Gated on viable cells.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

ab315376 staining phosphorylated Parkin (S65). HeLa cell lines with WT Parkin or S65A mutant overexpression (kindly provided by the Muqit lab, for details see PMID 26116755) were treated with 10 µM antimycin A and 1 µM oligomycin for 2 h. DMSO was used as control. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab315376 at 0.016 µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150083, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor®647), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor®594), pre-adsorbed at 1/1000 dilution (shown in magenta). Nuclear DNA was labelled with DAPI (shown in blue). Staining is seen in A+O treated HeLa WT Parkin cells, but not in the other conditions. Absence of staining in the HeLa S65A Parkin cells indicates phospho-specificity towards residue S65.

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Human PBMC (human peripheral blood mononuclear cell) cells labelling Granzyme A with ab321992 at 1/50 dilution (1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Cells were co-stained with anti human CD14 conjugated to Pacific blue.

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of Human PBMC (human peripheral blood mononuclear cell) cells labelling CD101 with ab325180 at 1/50 dilution (1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Cells were co-stained with CD11c conjugated to Alexa Fluor®488.
Gated on viable cells.

• Flow Cyt

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of Human purified neutrophils cells labelling IL-17RA Receptor with ab325606 at 1/500 dilution (0.1ug) / Red compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.

• Flow Cyt

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of Human PBMC (human peripheral blood mononuclear cell) cells labelling CD101 with ab325180 at 1/50 dilution (1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Cells were co-stained with CD8a conjugated to Pacific Blue.
Gated on viable cells.

• Flow Cyt

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of Human PBMC (human peripheral blood mononuclear cell) cells labelling CD101 with ab325180 at 1/50 dilution (1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Cells were co-stained with CD56 conjugated to PE.
Gated on viable cells.

• Flow Cyt

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of Human PBMC (human peripheral blood mononuclear cell) cells labelling CD1d with ab324952 at 1/500 dilution (0.1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Cells were co-stained with anti-CD19 conjugated to PE/CY7.

• Flow Cyt

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of Parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell) (Green and black) IL17RA KO HAP1(HO010217) (Magenta and grey) cells labelling IL-17RA Receptor with ab325606 at 1/500 dilution (0.1ug) / Magenta and Green compared with a Rabbit monoclonal IgG (ab172730) / Black and Grey isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.

• Flow Cyt

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of Human PBMC (human peripheral blood mononuclear cell) cells labelling CD101 with ab325180 at 1/50 dilution (1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Cells were co-stained with CD4 conjugated to Brilliant Violet 421.
Gated on viable cells.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Intracellular Flow Cytometry analysis of HEK-293T (Human embryonic kidney epithelial cell) cells transfected with a SARS-CoV-2 orf8 (Left) expression vector containing a myc tag or SARS-CoV2 orf3d (Right) expression vector containing GFP tag labeling SARS-CoV-2 orf3d with ab284040 at 1/500 dilution (Left and Right). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 647, ab150083) secondary antibody was used at 1/5000 dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

ab315377 staining phosphorylated Parkin (S65). HeLa cell lines with WT Parkin or S65A mutant overexpression (kindly provided by the Muqit lab, for details see PMID 26116755) were treated with 10 µM antimycin A and 1 µM oligomycin for 2 h. DMSO was used as control. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab315377 at 0.016 µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150083, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in magenta). Nuclear DNA was labelled with DAPI (shown in blue). Staining is seen in A+O treated HeLa WT Parkin cells, but not in the other conditions. Absence of staining in the HeLa S65A Parkin cells indicates phospho-specificity towards residue S65.

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

ab315377 staining phosphorylated Parkin (S65) and the staining co-localises with mitochondrial marker (Anti-COX IV antibody, ab33985). HeLa cell line with WT Parkin overexpression (kindly provided by the Muqit lab, for details see PMID 26116755) were treated with 10 µM antimycin A and 1 µM oligomycin for 2 h. DMSO was used as control. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab315377 at 0.08 µg/ml and ab33985, Mouse monoclonal Anti-COX IV antibody [mAbcam33985]. Cells were then incubated with ab150083, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in magenta). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Intracellular Flow Cytometry analysis of HEK-293T (Human embryonic kidney epithelial cell) cells transfected with a SARS-CoV-2 nsp9 (Left) or SARS-CoV2 nsp1 (Right) expression vector containing a myc tag labeling SARS-COV-2 nsp1 with ab284631 at 1/5000 dilution (left and right). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647, ab150083) secondary antibody was used at 1/5000.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype (upper left) / 293T (human embryonic kidney epithelial cell) cells transfected with an empty expression vector containing a Myc tag (upper middle) / 293T cells transfected with a IFITM1 expression vector containing a GFP tag (down left) / 293T cells transfected with a IFITM2 expression vector containing a GFP tag (down middle) / 293T cells transfected with a IFITM3 expression vector containing a GFP tag(down right) cells labelling IFITM2+IFITM3 with ab323747 at 1/5000 dilution (0.01 ug) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

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Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of Isotype (Left) / 293T cells transfected with a human SIGLEC6 expression vector containing a GFP-tag® (Middle) / 293T cells transfected with an empty expression vector containing a GFP-tag® (Right) cells labelling SIGLEC6 with ab323795 at 1/5000 dilution (0.01 ug).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

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Supplier Data

Flow Cytometry - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of Isotype (Left) / 293T cells transfected with a SIGLEC6 expression vector containing a GFP-tag (Middle) /293T cells transfected an empty vector containing a GFP-tag (Right) cells labelling SIGLEC6 with ab317309 at 1/5000 dilution (0.01ug) / Middle and Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control.

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed (AB150083)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Raji (human Burkitt's lymphoma B lymphocyte, Left) / HDLM-2 (human Hodgkin lymphoma cell, Right) cells labelling CISH/CIS with ab324909 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Negative control : Raji (PMID : 11032736).