Peripheral blood mononuclear cells were incubated with antibodies against CD3 (UCHT1) and CD28 (CD28.2) (all from Biolegend) to stimulate T cells, CD16 (3G8) (Biolegend) to stimulate NK cells, or F(ab′)2 anti-human IgM + IgG (eBioscience) to stimulate B cells in the presence of DMSO or inhibitors for 30 min on ice. Cells were washed and incubated with secondary Goat anti-mouse antibodies for 15 min, followed by stimulation for 2 min at 37°C. Cells were then fixed by BD Phosflow Fixation buffer and stained for extracellular markers with CD19-AF700 (HIB19), CD56-BV510 (NCAM16.2) and CD3-PerCP (UCHT1) (all from Biolegend). Cells were then permeabilized by BD Perm 2, stained with p-SLP-76 Y128 AF647 (J141-668.36.58) or p-SLP-65 Y84 AF647 (J117-1278) (both from BD) and analyzed by flow cytometry on a BD LSRFortessa.
Cd3 percp ucht1
Manufactured by BioLegend
CD3-PerCP (UCHT1) is a fluorochrome-conjugated antibody that binds to the CD3 antigen on human T cells. It is designed for use in flow cytometry applications to identify and enumerate T cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Negative selection microbeads kit, by Miltenyi Biotec (2 mentions)
Cd4 okt4 bv510, by BioLegend (2 mentions)
Cd8 rpa t8 fitc, by BioLegend (2 mentions)
Facsmelody, by BD (2 mentions)
Phosflow fixation buffer, by BD (1 mentions)
Cd28 cd28.2, by BioLegend (1 mentions)
Perm2, by BD (1 mentions)
Cd16 3g8, by BioLegend (1 mentions)
1 step fix lyse solution, by Thermo Fisher Scientific (1 mentions)
Cd226 pe, by BioLegend (1 mentions)
Cd56 bv605 hcd56, by BioLegend (1 mentions)
Cd16 fitc 3gb, by Beckman Coulter (1 mentions)
Cd25 bv421 m a251, by BD (1 mentions)
Nkg2a fitc rea110, by Miltenyi Biotec (1 mentions)
Anti ifnγ pe b27, by BioLegend (1 mentions)
Cd56 pe cy7 hcd56, by BioLegend (1 mentions)
Facsaria 2, by BD (1 mentions)
Golgistop, by BD (1 mentions)
6 protocols using cd3 percp ucht1
1
Phospho-flow analysis of immune cell signaling
2
Multiparametric Flow Cytometry Profiling
3
CLL Cell Cytotoxicity Assay with IL-15-Activated PBMC
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Healthy donor PBMC were incubated with 1 ng/mL IL-15 overnight and subsequently co-cultured with selinexor- or leptomycin B-treated CLL cells at an E:T ratio of 1:1 for 4 h at 37 °C. 0.17 µg/mL α-CD107a-eFluor660 (eBioH4A3, Invitrogen) was added to PBMCs and golgiStop (BD Biosciences) added after 1 h. PBMC were then stained with the following antibodies in FACS buffer (PBS, BSA 1%, Sodium Azide 0.05%) at 4 °C for 30 min: CD3-PerCP (UCHT1, Biolegend), CD56-PE/Cy7 (hCD56, Biolegend) and NKG2A-FITC (REA110, Miltenyi Biotech). Cells were then permeabilized and fixed with BD Cytofix/Cytoperm (BD Biosciences), stained with anti-IFNγ-PE (B27, Biolegend) and assessed using a BD FACS Aria II.
4
Sorting CD4+ Memory T Cells
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Previously frozen PBMCs were thawed at 37°C and washed once with RPMI 10% fetal calf serum and twice with RPMI 5% fetal calf serum for sorting. Approximately 5 million PBMCs were labeled at 4°C for 30 min with the following monoclonal antibody (mAbs) mix: CD8(RPTA-T8)-FITC, CD3(UCHT1)-PerCP, CD4(RPA-T4)-APC/Cyanine7, CD27(O323)-Brilliant Violet 765, CD45RO(UCHL1)-PE (All Biolegend). After the staining was finished, samples were split in two portions and sorted separately to obtain duplicates. CD4+ memory T cells were defined as CD27+/CD45RO+, CD27-/CD45RO+, or CD27-/CD45RO- (thus, all CD4+ cells, except the naive T cells (CD27+/CD45RO-)) and sorted using a FACS Melody cell sorter (BD). CD4+ memory T cells were sorted directly into PBS, spun down and resuspended in RNA Later (Ambion Inc. Applied Biosystems). Sorted samples were stored at −80°C for subsequent TCRβ clonotype analysis.
5
Isolating and Characterizing CMV-Specific CD8+ T Cells
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CD8+ T cells were isolated from PBMCs using a negative selection microbeads kit (Miltenyi Biotec) according to the manufacturer's protocol. Next, CD8+ T cells were labeled at room temperature for 20 min with the A*0201/GLCTLVAML-APC dextramer and with the A*0201/NLVPMVATV-APC dextramer for CMV+ individuals. Subsequently surface staining was performed using the following mAbs: CD3(UCHT1)-PerCP (Biolegend), CD4(OKT4)-BV510 (Biolegend), and CD8(RPA-T8)-FITC (Biolegend). CD3+CD4−CD8+dextramer+ cells were then sorted by FACS Melody (BD) directly into RNAlater (Ambion Inc. Applied Biosystems) and stored at −80°C for subsequent TCRβ clonotype analysis.
6
Isolation and Characterization of GILG-Specific CD8+ T Cells
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CD8+ T cells were isolated from PBMCs using a negative selection microbeads kit (Miltenyi Biotec). Next, CD8+ T cells were labeled at room temperature for 20 minutes with the A*0201/GILGFVFTL-APC dextramer (Immudex) (GILG). Subsequently surface staining was added with the following mAbs: CD3(UCHT1)-PerCP (Biolegend), CD4(OKT4)-BV510 (Biolegend) and CD8(RPA-T8)-FITC (Biolegend) and CD3+CD4-CD8+GILG+ cells were sorted by FACS Melody (BD) directly into RNAlater (Ambion Inc. Applied Biosystems) and stored at -80°C for subsequent TCRβ clonotype analysis.
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