Cd8b 2st8.5h7

Manufactured by Beckman Coulter

CD8b (2ST8.5H7) is a monoclonal antibody that binds to the CD8 beta chain, a component of the CD8 co-receptor expressed on the surface of certain T lymphocytes. This antibody is designed for use in flow cytometry applications to identify and quantify CD8-positive cells within a sample.

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4 protocols using cd8b 2st8.5h7

Flow Cytometric Profiling of PBMCs

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Frozen PBMCs were thawed, washed in FACS buffer (2% FBS, 1mM EDTA in PBS), and counted on TC20 (Biorad) before surface staining using two independent flow panels. For the innate panel, the following antibodies were used: CD3 (SP34, BD Pharmingen) and CD20 (2H7, BioLegend) for the exclusion of T & B lymphocytes, respectively. We further stained for CD56 (HCD56, Biolegend), CD57 (HNK-1, BioLegend), KLRG1 (SA231A2, BioLegend) CD16 (3G8, BioLegend), CD14 (M5E2, BioLegend), HLA-DR (L243, BioLegend), CD11c (3.9, ThermoFisher Scientific), CD123 (6H6, BioLegend) and CD86 (IT2.2, BioLegend). For the adaptive panel, the following antibodies were used: CD4 (OKT4, BioLegend), CD8b (2ST8.5H7, Beckman Coulter), CD45RA (HI100, TONBO Biosciences), CCR7 (G043H7, BD Biosciences), CD19 (HIB19, BioLegend), IgD (IA6–2, BioLegend), CD27 (M-T271, BioLegend), KLRG1 (SA231A2, BioLegend) and PD-1 (Eh12.2h7, BioLegend). Cells were stained with Ghost Dye viability dye (TONBO biosciences) for 30 minutes at 4C per manufacturer’s instructions, washed, surface stained with either innate or adaptive panels for 30 minutes at 4C. Samples were then washed and analyzed on Attune NxT Flow Cytometer (ThermoFisher Scientific, Waltham MA).

Sureshchandra S., Zulu M.Z., Doratt B., Jankeel A., Tifrea D., Edwards R., Rincon M., Marshall N.E, & Messaoudi I. (2021). Deep immune profiling of the maternal-fetal interface with mild SARS-CoV-2 infection. bioRxiv.

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Immunophenotyping of Frozen PBMCs

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Frozen PBMCs were thawed, washed in FACS buffer (2% FBS, 1 mM EDTA in PBS), and counted on TC20 (Biorad) before surface staining using two independent flow panels. For the innate panel, the following antibodies were used: CD3 (SP34, BD Pharmingen) and CD20 (2H7, BioLegend) for the exclusion of T & B lymphocytes, respectively. We further stained for CD56 (HCD56, Biolegend), CD57 (HNK-1, BioLegend), KLRG1 (SA231A2, BioLegend) CD16 (3G8, BioLegend), CD14 (M5E2, BioLegend), HLA-DR (L243, BioLegend), CD11c (3.9, ThermoFisher Scientific), CD123 (6H6, BioLegend) and CD86 (IT2.2, BioLegend). For the adaptive panel, the following antibodies were used: CD4 (OKT4, BioLegend), CD8b (2ST8.5H7, Beckman Coulter), CD45RA (HI100, TONBO Biosciences), CCR7 (G043H7, Biolegend), CD19 (HIB19, BioLegend), IgD (IA6-2, BioLegend), CD27 (M-T271, BioLegend), KLRG1 (SA231A2, BioLegend) and PD-1 (Eh12.2h7, BioLegend). Cells were stained with Ghost Dye viability dye (TONBO biosciences) for 30 min at 4C per manufacturer’s instructions, washed, surface stained with either innate or adaptive panels for 30 min at 4C. Samples were then washed and analyzed on Attune NxT Flow Cytometer (ThermoFisher Scientific, Waltham MA).

Sureshchandra S., Zulu M.Z., Doratt B.M., Jankeel A., Tifrea D., Edwards R., Rincon M., Marshall N.E, & Messaoudi I. (2022). Single-cell RNA sequencing reveals immunological rewiring at the maternal-fetal interface following asymptomatic/mild SARS-CoV-2 infection. Cell Reports, 39(11), 110938.

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Multiparametric Phenotyping of Decidual Leukocytes

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1-2 X 10⁶ fresh decidual leukocytes were washed with PBS and stained using the following cocktail of antibodies: CD45 (HI30, Biolegend), CD66b (G10F5, BioLegend), CD20 (2H7, BioLegend), CD4 (OKT4, BioLegend), CD8b (2ST8.5H7, Beckman Coulter), CD14 (M5E2, BioLegend), HLA-DR (L243, BioLegend), CD56 (HCD56, Biolegend), CD16 (3G8, BioLegend), CD11c (3.9, ThermoFisher Scientific), CD123 (6H6, BioLegend), for 20 min in dark at 4C. Samples were washed twice in FACS buffer and resuspended in 400 uL. All samples were acquired with the Attune NxT Flow Cytometer (ThermoFisher Scientific, Waltham MA), immediately after addition of SYTOX Red Dead Cell Stain (1:1000). Data were analyzed using FlowJO (Ashland OR).

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Comprehensive Immune Profiling of Decidual Leukocytes

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1–2 × 10⁶ fresh decidual leukocytes were washed with PBS and stained using the following cocktail of antibodies: CD45 (HI30, Biolegend), CD66b (G10F5, BioLegend), CD20 (2H7, BioLegend), CD4 (OKT4, BioLegend), CD8b (2ST8.5H7, Beckman Coulter), CD14 (M5E2, BioLegend), HLA-DR (L243, BioLegend), CD56 (HCD56, Biolegend), CD16 (3G8, BioLegend), CD11c (3.9, ThermoFisher Scientific), CD123 (6H6, BioLegend), for 20 minutes in dark at 4C. Samples were washed twice in FACS buffer and resuspended in 400 uL. All samples were acquired with the Attune NxT Flow Cytometer (ThermoFisher Scientific, Waltham MA), immediately after addition of SYTOX Red Dead Cell Stain (1:1000). Data were analyzed using FlowJO (Ashland OR).

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