Human Monocyte STING Activation Flow Cytometry Panel #69210
Product Information
Protocol
Product Description
The Human Monocyte STING Activation Flow Cytometry Panel includes antibodies targeting phospho-STING (Ser366) and phenotyping markers to enable observation of STING activation in human monocyte populations among peripheral blood mononuclear cells (PBMCs).
CD45 is a pan leukocyte marker. Monocytes and dendritic cells (DCs) are identified by co-expression of CD11b and HLA-DR. Monocytes can be distinguished from DCs by expression of CD14. Classical monocytes are CD14+CD16-, intermediate monocytes are CD14+CD16+, and nonclassical monocytes are CD14dimCD16+. DCs are CD14-CD16-. STING mediates the innate immune response to DNA. STING is activated upon binding of the second messenger cGAMP, which is produced by cGAS. Binding of cGAMP to cGAS triggers conformational changes in STING, trafficking of STING from the ER to the Golgi, and recruitment of TBK1, which phosphorylates STING at Ser366.
CD45 is a pan leukocyte marker. Monocytes and dendritic cells (DCs) are identified by co-expression of CD11b and HLA-DR. Monocytes can be distinguished from DCs by expression of CD14. Classical monocytes are CD14+CD16-, intermediate monocytes are CD14+CD16+, and nonclassical monocytes are CD14dimCD16+. DCs are CD14-CD16-. STING mediates the innate immune response to DNA. STING is activated upon binding of the second messenger cGAMP, which is produced by cGAS. Binding of cGAMP to cGAS triggers conformational changes in STING, trafficking of STING from the ER to the Golgi, and recruitment of TBK1, which phosphorylates STING at Ser366.
Product Usage Information
STING activation can be induced through treatment with a variety of STING agonists including Poly(dA:dT) Sodium Salt #47945 and the second messenger 2',3'-cGAMP (sodium salt) #35573. All antibodies in this kit are compatible with the Intracellular Flow Cytometry Kit (Triton X-100) #51995 and can be used in a single staining mix on fixed and permeabilized cells. Prior to fixation and antibody incubation, we recommend adding a fixable viability dye such as the Ghost Dye Violet 510 Fixable Viability Dye #59863 to enable identification and exclusion of dead cells from analysis.
For individual product dilutions, refer to panel product datasheet or individual product pages.
Gating strategy for observing STING phosphorylation in monocyte populations: If a fixable viability dye was used, first gate on viable cells. Next, gate on CD45+ immune cells. Viewing the CD45+ population, gate on singlets using appropriate scatter parameters, such as FSC-A vs. FSC-H. Observe CD11b vs. HLA-DR expression and gate on CD11b+HLA-DR+ monocytes and DCs. Observe CD14 vs. CD16 on the gated population. Classical monocytes are CD14+CD16-, intermediate monocytes are CD14+CD16+, nonclassical monocytes are CD14dimCD16+, and DCs are CD14-CD16-. Monocytic MDSCs are CD11b+HLA-DR-CD14+ and can also be observed. Observe phospho-STING (Ser366) in populations of interest.
For individual product dilutions, refer to panel product datasheet or individual product pages.
Gating strategy for observing STING phosphorylation in monocyte populations: If a fixable viability dye was used, first gate on viable cells. Next, gate on CD45+ immune cells. Viewing the CD45+ population, gate on singlets using appropriate scatter parameters, such as FSC-A vs. FSC-H. Observe CD11b vs. HLA-DR expression and gate on CD11b+HLA-DR+ monocytes and DCs. Observe CD14 vs. CD16 on the gated population. Classical monocytes are CD14+CD16-, intermediate monocytes are CD14+CD16+, nonclassical monocytes are CD14dimCD16+, and DCs are CD14-CD16-. Monocytic MDSCs are CD11b+HLA-DR-CD14+ and can also be observed. Observe phospho-STING (Ser366) in populations of interest.
Storage
CD45 (HI30) Mouse mAb (violetFluor 450 Conjugate), CD11b/ITGAM (M1/70) Rat mAb (FITC Conjugate), HLA-DR (L243) Mouse mAb (PerCP Conjugate), CD14 (61D3) Mouse mAb (redFluor 710 Conjugate), and CD16 (3G8) Mouse mAb (PE Conjugate) are supplied in 10 mM NaH2PO4, 150 mM NaCl, 0.09% NaN3, 0.1% gelatin, pH 7.2. Phospho-STING (Ser366) (D8K6H) Rabbit mAb (Alexa Fluor® 647 Conjugate) is supplied in PBS (pH 7.2), less than 0.1% sodium azide, and 2 mg/mL BSA. Store at 4oC. Do not aliquot the antibodies. Protect from light. Do not freeze.
All components in this kit are stable in accordance with the date printed on the outer packaging label when stored at the recommended temperature. Please refer to product labels, datasheets, or web pages for specific “Best By” dates for each individual component.
All components in this kit are stable in accordance with the date printed on the outer packaging label when stored at the recommended temperature. Please refer to product labels, datasheets, or web pages for specific “Best By” dates for each individual component.
Specificity / Sensitivity
Each antibody in the Human Monocyte STING Activation Flow Cytometry Panel detects endogenous levels of its target protein. CD45 (HI30) Mouse mAb (violetFluor 450 Conjugate), CD11b/ITGAM (M1/70) Rat mAb (FITC Conjugate), HLA-DR (L243) Mouse mAb (PerCP Conjugate), CD14 (61D3) Mouse mAb (redFluor 710 Conjugate), and CD16 (3G8) Mouse mAb (PE Conjugate) detect epitopes within the extracellular domain. Phospho-STING (Ser366) (D8K6H) Rabbit mAb (Alexa Fluor® 647 Conjugate) recognizes endogenous levels of STING protein only when phosphorylated at Ser366.
Species Reactivity:
Human
Source / Purification
Monoclonal antibodies were purified from tissue culture supernatant via affinity chromatography. The purified antibodies were conjugated under optimal conditions, with unreacted dye removed from the preparation.
限制使用
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