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A. Identification of human microglial cell surface proteins and panel design. For each of the datasets (generated proteomics dataset from freshly isolated human microglia, microglia-specific disease-relevant GWAS hits, Input ), cell surface and cell membrane proteins were identified using the databases Uniprot ( https://www.uniprot.org/ ) and the Surfaceome ( Query ). Proteins/candidates that scored in both approaches - the Uniprot and Surfaceome analysis - for the proteomics as well as the GWAS hits, were subsequently compiled into a list of n=91 initial candidates ( Output ). Candidate expression was then assessed in other available proteomics datasets of HCM3 microglia and iPSC-derived microglia as well as brain RNA-Seq datasets (n=87 candidates), commercially available antibodies were then titrated using flow cytometry (n=32) and selected candidates subsequently added to the production of the customized microglia-specific CITE-Seq panel produced in Total-Seq A (TS-A) format by BioLegend (n=17 microglia-specific antibodies, n=150 Universal cocktail 1.0 TS-A antibodies)( Panel assembly ). B. Examples for titration results for HMC3 microglia. To evaluate all titration results for the antibody candidates titrated in HMC3 microglia, the PE-positive population of all samples (unstained 1 , Isotype control 2 , 1:32 3 , 1:16 4 , 1:8 5 , 1:4 6 , 1:2 7 , recommended concentration 8 ) were concatenated and plotted based on their Comp-PE-A values. For visualization, ADAM10 and <t>PLXNB2</t> are shown here. C. Examples for titration results for PBMCs. Examples for titration results for PBMCs. To evaluate all titration results for the antibody candidates titrated in PBMCs, the PE-positive population of all samples (unstained 1 , Isotype control 2 , 1:32 3 , 1:16 4 , 1:8 5 , 1:4 6 , 1:2 7 , recommended concentration 8 , doubled recommended concentration (2R) 9 ) gated on lymphocytes and monocytes were concatenated and plotted based on their Comp-PE-A values. For visualization, ADAM10 and PLXNB2 are shown here.
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A. Identification of human microglial cell surface proteins and panel design. For each of the datasets (generated proteomics dataset from freshly isolated human microglia, microglia-specific disease-relevant GWAS hits, Input ), cell surface and cell membrane proteins were identified using the databases Uniprot ( https://www.uniprot.org/ ) and the Surfaceome ( Query ). Proteins/candidates that scored in both approaches - the Uniprot and Surfaceome analysis - for the proteomics as well as the GWAS hits, were subsequently compiled into a list of n=91 initial candidates ( Output ). Candidate expression was then assessed in other available proteomics datasets of HCM3 microglia and iPSC-derived microglia as well as brain RNA-Seq datasets (n=87 candidates), commercially available antibodies were then titrated using flow cytometry (n=32) and selected candidates subsequently added to the production of the customized microglia-specific CITE-Seq panel produced in Total-Seq A (TS-A) format by BioLegend (n=17 microglia-specific antibodies, n=150 Universal cocktail 1.0 TS-A antibodies)( Panel assembly ). B. Examples for titration results for HMC3 microglia. To evaluate all titration results for the antibody candidates titrated in HMC3 microglia, the PE-positive population of all samples (unstained 1 , Isotype control 2 , 1:32 3 , 1:16 4 , 1:8 5 , 1:4 6 , 1:2 7 , recommended concentration 8 ) were concatenated and plotted based on their Comp-PE-A values. For visualization, ADAM10 and <t>PLXNB2</t> are shown here. C. Examples for titration results for PBMCs. Examples for titration results for PBMCs. To evaluate all titration results for the antibody candidates titrated in PBMCs, the PE-positive population of all samples (unstained 1 , Isotype control 2 , 1:32 3 , 1:16 4 , 1:8 5 , 1:4 6 , 1:2 7 , recommended concentration 8 , doubled recommended concentration (2R) 9 ) gated on lymphocytes and monocytes were concatenated and plotted based on their Comp-PE-A values. For visualization, ADAM10 and PLXNB2 are shown here.
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A. Identification of human microglial cell surface proteins and panel design. For each of the datasets (generated proteomics dataset from freshly isolated human microglia, microglia-specific disease-relevant GWAS hits, Input ), cell surface and cell membrane proteins were identified using the databases Uniprot ( https://www.uniprot.org/ ) and the Surfaceome ( Query ). Proteins/candidates that scored in both approaches - the Uniprot and Surfaceome analysis - for the proteomics as well as the GWAS hits, were subsequently compiled into a list of n=91 initial candidates ( Output ). Candidate expression was then assessed in other available proteomics datasets of HCM3 microglia and iPSC-derived microglia as well as brain RNA-Seq datasets (n=87 candidates), commercially available antibodies were then titrated using flow cytometry (n=32) and selected candidates subsequently added to the production of the customized microglia-specific CITE-Seq panel produced in Total-Seq A (TS-A) format by BioLegend (n=17 microglia-specific antibodies, n=150 Universal cocktail 1.0 TS-A antibodies)( Panel assembly ). B. Examples for titration results for HMC3 microglia. To evaluate all titration results for the antibody candidates titrated in HMC3 microglia, the PE-positive population of all samples (unstained 1 , Isotype control 2 , 1:32 3 , 1:16 4 , 1:8 5 , 1:4 6 , 1:2 7 , recommended concentration 8 ) were concatenated and plotted based on their Comp-PE-A values. For visualization, ADAM10 and <t>PLXNB2</t> are shown here. C. Examples for titration results for PBMCs. Examples for titration results for PBMCs. To evaluate all titration results for the antibody candidates titrated in PBMCs, the PE-positive population of all samples (unstained 1 , Isotype control 2 , 1:32 3 , 1:16 4 , 1:8 5 , 1:4 6 , 1:2 7 , recommended concentration 8 , doubled recommended concentration (2R) 9 ) gated on lymphocytes and monocytes were concatenated and plotted based on their Comp-PE-A values. For visualization, ADAM10 and PLXNB2 are shown here.
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A. Identification of human microglial cell surface proteins and panel design. For each of the datasets (generated proteomics dataset from freshly isolated human microglia, microglia-specific disease-relevant GWAS hits, Input ), cell surface and cell membrane proteins were identified using the databases Uniprot ( https://www.uniprot.org/ ) and the Surfaceome ( Query ). Proteins/candidates that scored in both approaches - the Uniprot and Surfaceome analysis - for the proteomics as well as the GWAS hits, were subsequently compiled into a list of n=91 initial candidates ( Output ). Candidate expression was then assessed in other available proteomics datasets of HCM3 microglia and iPSC-derived microglia as well as brain RNA-Seq datasets (n=87 candidates), commercially available antibodies were then titrated using flow cytometry (n=32) and selected candidates subsequently added to the production of the customized microglia-specific CITE-Seq panel produced in Total-Seq A (TS-A) format by BioLegend (n=17 microglia-specific antibodies, n=150 Universal cocktail 1.0 TS-A antibodies)( Panel assembly ). B. Examples for titration results for HMC3 microglia. To evaluate all titration results for the antibody candidates titrated in HMC3 microglia, the PE-positive population of all samples (unstained 1 , Isotype control 2 , 1:32 3 , 1:16 4 , 1:8 5 , 1:4 6 , 1:2 7 , recommended concentration 8 ) were concatenated and plotted based on their Comp-PE-A values. For visualization, ADAM10 and <t>PLXNB2</t> are shown here. C. Examples for titration results for PBMCs. Examples for titration results for PBMCs. To evaluate all titration results for the antibody candidates titrated in PBMCs, the PE-positive population of all samples (unstained 1 , Isotype control 2 , 1:32 3 , 1:16 4 , 1:8 5 , 1:4 6 , 1:2 7 , recommended concentration 8 , doubled recommended concentration (2R) 9 ) gated on lymphocytes and monocytes were concatenated and plotted based on their Comp-PE-A values. For visualization, ADAM10 and PLXNB2 are shown here.
Pe Anti Plxnb2, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+plxnb2/Plexin-B2+Antibody%2C+anti-human%2C+PE%2C+REAfinity/pm38025776-230-42-44
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A. Identification of human microglial cell surface proteins and panel design. For each of the datasets (generated proteomics dataset from freshly isolated human microglia, microglia-specific disease-relevant GWAS hits, Input ), cell surface and cell membrane proteins were identified using the databases Uniprot ( https://www.uniprot.org/ ) and the Surfaceome ( Query ). Proteins/candidates that scored in both approaches - the Uniprot and Surfaceome analysis - for the proteomics as well as the GWAS hits, were subsequently compiled into a list of n=91 initial candidates ( Output ). Candidate expression was then assessed in other available proteomics datasets of HCM3 microglia and iPSC-derived microglia as well as brain RNA-Seq datasets (n=87 candidates), commercially available antibodies were then titrated using flow cytometry (n=32) and selected candidates subsequently added to the production of the customized microglia-specific CITE-Seq panel produced in Total-Seq A (TS-A) format by BioLegend (n=17 microglia-specific antibodies, n=150 Universal cocktail 1.0 TS-A antibodies)( Panel assembly ). B. Examples for titration results for HMC3 microglia. To evaluate all titration results for the antibody candidates titrated in HMC3 microglia, the PE-positive population of all samples (unstained 1 , Isotype control 2 , 1:32 3 , 1:16 4 , 1:8 5 , 1:4 6 , 1:2 7 , recommended concentration 8 ) were concatenated and plotted based on their Comp-PE-A values. For visualization, ADAM10 and PLXNB2 are shown here. C. Examples for titration results for PBMCs. Examples for titration results for PBMCs. To evaluate all titration results for the antibody candidates titrated in PBMCs, the PE-positive population of all samples (unstained 1 , Isotype control 2 , 1:32 3 , 1:16 4 , 1:8 5 , 1:4 6 , 1:2 7 , recommended concentration 8 , doubled recommended concentration (2R) 9 ) gated on lymphocytes and monocytes were concatenated and plotted based on their Comp-PE-A values. For visualization, ADAM10 and PLXNB2 are shown here.

Journal: bioRxiv

Article Title: A proteogenomic tool uncovers protein markers for human microglial states

doi: 10.1101/2025.03.31.646212

Figure Lengend Snippet: A. Identification of human microglial cell surface proteins and panel design. For each of the datasets (generated proteomics dataset from freshly isolated human microglia, microglia-specific disease-relevant GWAS hits, Input ), cell surface and cell membrane proteins were identified using the databases Uniprot ( https://www.uniprot.org/ ) and the Surfaceome ( Query ). Proteins/candidates that scored in both approaches - the Uniprot and Surfaceome analysis - for the proteomics as well as the GWAS hits, were subsequently compiled into a list of n=91 initial candidates ( Output ). Candidate expression was then assessed in other available proteomics datasets of HCM3 microglia and iPSC-derived microglia as well as brain RNA-Seq datasets (n=87 candidates), commercially available antibodies were then titrated using flow cytometry (n=32) and selected candidates subsequently added to the production of the customized microglia-specific CITE-Seq panel produced in Total-Seq A (TS-A) format by BioLegend (n=17 microglia-specific antibodies, n=150 Universal cocktail 1.0 TS-A antibodies)( Panel assembly ). B. Examples for titration results for HMC3 microglia. To evaluate all titration results for the antibody candidates titrated in HMC3 microglia, the PE-positive population of all samples (unstained 1 , Isotype control 2 , 1:32 3 , 1:16 4 , 1:8 5 , 1:4 6 , 1:2 7 , recommended concentration 8 ) were concatenated and plotted based on their Comp-PE-A values. For visualization, ADAM10 and PLXNB2 are shown here. C. Examples for titration results for PBMCs. Examples for titration results for PBMCs. To evaluate all titration results for the antibody candidates titrated in PBMCs, the PE-positive population of all samples (unstained 1 , Isotype control 2 , 1:32 3 , 1:16 4 , 1:8 5 , 1:4 6 , 1:2 7 , recommended concentration 8 , doubled recommended concentration (2R) 9 ) gated on lymphocytes and monocytes were concatenated and plotted based on their Comp-PE-A values. For visualization, ADAM10 and PLXNB2 are shown here.

Article Snippet: Antibodies that were successfully titrated in HMC3s included: ADAM10 (BioLegend, Cat#: 352703), CD49 (BioLegend, Cat#: 328009), CD51 (BioLegend, Cat#: 327909), HLA-C (BioLegend, Cat#: 373302), ITGB5 (BioLegend, Cat#: 345203), PLXNB2 (Miltenyi, Cat#: 130-095-212), PTPRJ (BioLegend, Cat#: 328708).

Techniques: Generated, Isolation, Membrane, Expressing, Derivative Assay, RNA Sequencing, Flow Cytometry, Produced, Titration, Control, Concentration Assay