Surface staining was carried out using PBS with 1% FBS. For intracellular staining of cytokines, cells were first surface stained, then fixed with 1% paraformaldehyde (catalog no. 19943; Affymetrix, Santa Clara, CA) for 15 min at room temperature and permeabilized in permeabilization buffer (TNB-1213-L150; Tonbo Biosciences) before staining for cytokines. Staining for transcription factors was performed with the Foxp3/Transcription Factor Staining Buffer Kit (TNB-1022-L160; Tonbo Biosciences) according to the manufacturer’s instructions. Flow cytometry data were collected with CytoFLEX (BD Biosciences, Franklin Lake, NJ), and results were analyzed using FlowJo software (Becton Dickinson, Franklin Lakes, NJ). Abs specific for mouse CD4 (RM4–5), CD8 (53–6.7), and IL-4 (11B11) were purchased from Tonbo Biosciences; and those for IFN-γ (4S.B3), TNF (MAB11), GATA3 (L50–823), and Tbet (eBio4B10) were purchased from eBioscience (San Diego, CA). Abs specific for human CD4 (RPA-T4), CD8 (RPA-T8), and IL-4 (MP4–25D2) were purchased from Tonbo Biosciences; and those for IFN-γ (4S.B3), GATA3 (16E10A23), and Tbet (eBio4B10) were obtained from eBioscience.
Ebio4b10
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EBio4B10 is a laboratory instrument designed for electrophoresis applications. It offers precise control over voltage, current, and power settings to enable consistent and reliable electrophoresis experiments.
Automatically generated - may contain errors
Lab products found in correlation
Fjk 16, by Thermo Fisher Scientific (3 mentions)
Foxp3 transcription factor staining buffer kit, by Cytek Biosciences (2 mentions)
Rm4 5, by Cytek Biosciences (2 mentions)
Rpa t4, by Cytek Biosciences (2 mentions)
Tnb 1213 l150, by Cytek Biosciences (2 mentions)
Rpa t8, by Cytek Biosciences (2 mentions)
Mp4 25d2, by Cytek Biosciences (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
Monensin, by Merck Group (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Afkjs 9, by Thermo Fisher Scientific (2 mentions)
Anti mouse ifn γ xmg1, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd4 rm4 5, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd45 30 f11, by Thermo Fisher Scientific (2 mentions)
Anti mouse il 17a tc11 18h10.1, by BioLegend (2 mentions)
Anti mouse foxp3 fjk 16s, by Thermo Fisher Scientific (2 mentions)
Golgiplug, by BD (2 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm buffer, by BD (2 mentions)
Zombie yellow fixable viability kit, by BioLegend (2 mentions)
12 protocols using ebio4b10
1
Comprehensive Cytokine Profiling with Flow Cytometry
2
Comprehensive Cytokine Profiling with Flow Cytometry
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Surface staining was carried out using PBS with 1% FBS. For intracellular staining of cytokines, cells were first surface stained, then fixed with 1% paraformaldehyde (catalog no. 19943; Affymetrix, Santa Clara, CA) for 15 min at room temperature and permeabilized in permeabilization buffer (TNB-1213-L150; Tonbo Biosciences) before staining for cytokines. Staining for transcription factors was performed with the Foxp3/Transcription Factor Staining Buffer Kit (TNB-1022-L160; Tonbo Biosciences) according to the manufacturer’s instructions. Flow cytometry data were collected with CytoFLEX (BD Biosciences, Franklin Lake, NJ), and results were analyzed using FlowJo software (Becton Dickinson, Franklin Lakes, NJ). Abs specific for mouse CD4 (RM4–5), CD8 (53–6.7), and IL-4 (11B11) were purchased from Tonbo Biosciences; and those for IFN-γ (4S.B3), TNF (MAB11), GATA3 (L50–823), and Tbet (eBio4B10) were purchased from eBioscience (San Diego, CA). Abs specific for human CD4 (RPA-T4), CD8 (RPA-T8), and IL-4 (MP4–25D2) were purchased from Tonbo Biosciences; and those for IFN-γ (4S.B3), GATA3 (16E10A23), and Tbet (eBio4B10) were obtained from eBioscience.
3
Intracellular Cytokine Staining and mTOR/STAT3 Activation
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For intracellular staining of IL-10, IL-17 and IFN-γ, cells were stained first for surface antigens with antibodies (e.g. RM4-5 for CD4 or 53-6.7 for CD8) and then activated in RPMI 1640 (10% FBS) with PMA (50 ng/ml), ionomycin (1 μM), and monensin (2 mM; Sigma-Aldrich) for 4 h. Cells were then fixed, permeabilized, and stained with antibodies to mIL-10 (JES5-16E3), mIL-17A (TC11-18H10.1) or IFN-γ (XMG1.2). Cells were stained with an antibody to mouse FoxP3 (FJK-16s), T-bet (eBio4B10), or RORγt (AFKJS-9) according to the manufacturer’s protocol (eBioscience). For co-staining of IL-10 and FoxP3, the cells prestained with anti-CD4 (RM4-5) were activated with PMA (50 ng/ml), ionomycin (1 μM), and Brefeldin A (25 μg/ml) for 4 h. Activated cells were fixed, permeabilized, and stained with antibodies to IL-10 and FoxP3.
For assessment of mTOR activation or STAT3 activation, mouse CD4+ cells were activated for up to 60 hours in complete RPMI-1640 medium with an immobilized antibody to CD3 (5 μg/ml) and soluble antibody to CD28 (2 μg/ml) in the presence of C2 (0, 1, 10, or 20 mM), C3 (0, 0.1, 0.5, or 1 mM) or C4 (0, 0.125, 0.25, or 0.5 mM). Antibodies to phosphorylated rS6 (Ser235/236; D57.2.2E), phosphorylated STAT3 (Y705; D3A7), phosphorylated MAPK (Erk1/2) (Thr202/Tyr204; 137F5) were used for flow cytometry (all from Cell Signaling Technology, Danvers, MA).
For assessment of mTOR activation or STAT3 activation, mouse CD4+ cells were activated for up to 60 hours in complete RPMI-1640 medium with an immobilized antibody to CD3 (5 μg/ml) and soluble antibody to CD28 (2 μg/ml) in the presence of C2 (0, 1, 10, or 20 mM), C3 (0, 0.1, 0.5, or 1 mM) or C4 (0, 0.125, 0.25, or 0.5 mM). Antibodies to phosphorylated rS6 (Ser235/236; D57.2.2E), phosphorylated STAT3 (Y705; D3A7), phosphorylated MAPK (Erk1/2) (Thr202/Tyr204; 137F5) were used for flow cytometry (all from Cell Signaling Technology, Danvers, MA).
4
Cytokine Expression Analysis by Flow Cytometry
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IL-17a and IFN-γ cytokine expression were assessed by flow cytometry as described previously45 (link). Briefly, the cells were stimulated for 4 h in phorbol 1,2-myristate 1,3-acetate (5 ng/ml) and ionomycin (1 µg/ml) in the presence of protein transport inhibitor GolgiPlug (BD Pharmingen). Cells were stained for surface markers with following antibodies: anti-mouse TCRβ (H57–597; eBioscience), anti-mouse CD4 (RM4–5; eBioscience), anti-mouse CD45 (30-F11, eBioscience). Cells were washed and fixed using Cytofix/Cytoperm buffer (BD Pharmingen or eBioscience). Intracellular staining was performed with following antibodies: anti-mouse IL-17a (TC11-18H10.1; BioLegend), anti-mouse IFN-γ (XMG1.2, eBiosciences), anti-mouse Foxp3 (FJK-16s, eBioscience) and anti-human/mouse T-bet (eBio4B10, eBiosciences). Dead cells were excluded from analysis using Zombie Yellow Fixable Viability Kit (Biolegend) or LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies).
5
Intracellular Cytokine Staining and mTOR/STAT3 Activation
6
Multiparametric Analysis of Immune Cell Subsets
7
Comprehensive Flow Cytometry Immunophenotyping
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Cells (1 × 106) were stained with FITC-, PE-, APC-, PerCP-, PECy7-, APC Cy7-, Pacific Blue-, or AmCyan-labeled monoclonal antibodies (mAbs) (BD Biosciences) to CD4 (H129.19 or GK1.5), CD19 (ID3), CD25 (PC61), CD39 (24DMS1), CD44 (IM7), CD62L (MEL-14), CD122 (TM-β1), CD178 (ICOS) (7E.17G9), CD127 (A7R34), GL7 (GL7), PD1 (J43), CXCR5 (2G8) and P2X7 (1F11). For the detection of intracellular staining, PE-labeled mAb to Bcl-6 (K112-91, BD Biosciences), PerCP-labeled mAb to T-bet (eBio4B10; eBioscience, USA), PE-labeled mAb to Blimp-1 (6D3; BD Biosciences) and APC-labeled mAb to Foxp3 (FJK-16s; eBioscience) were used according to the manufacturer’s instructions. PE-labeled rat IgG1 (BD Bioscience) and PerCP-labeled anti-CD45.1 mAb (A20; BD Bioscience) were used as isotype controls. Annexin V staining was performed in the appropriate binding buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2 [pH 7.4]). Cells were analyzed by flow cytometry using a FACSCanto device with DIVA software (BD Biosciences). Data were analyzed with FlowJo software v.7.2.2 (Tree Star Inc., USA).
8
Murine Melanoma Immunotherapy Protocols
9
Western Blot Analysis of T-bet Expression
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T-bet expression analysis by western blot was performed as described elsewhere.57 (link) Anti-T-bet antibody (eBio4B10, eBioscience) was used at a dilution of 1/1000. A primary antibody against β-actin (1/1000) (13E5, Cell Signalling Technology, Danvers, USA) was used as loading control. Peroxidase-conjugated anti-mouse IgG (1/5000) (GE Healthcare UK Limited) was used as secondary antibody. All antibody incubations were performed at 4 °C overnight.
10
Multiparameter Analysis of NK Cell Activation
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For analysis of surface markers CD71, CD98, and CD122, cells were stained in PBS containing 2% (wt/vol) FBS with antibodies from eBioscience or BD. Their expression level was presented as net mean fluorescence intensity (ΔMFI), which was determined by subtracting MFI of isotype control, or as fold relative to unstimulated, set as 1. Intracellular staining was used for NK cell transcription factor and phosphorylated proteins. NK cell transcription factors E4BP4, Eomes, and T-bet were stained with anti–mouse E4BP4 antibody (S2M-E19; eBioscience), anti-Eomes (Dan11mag; eBioscience), and anti–T-bet (eBio4B10; eBioscience) before and after overnight stimulation with IL-15–IL-15R complex according to the manufacturer’s instruction (eBioscience), similar to Foxp3 staining, respectively. For detection of phosphorylated signaling proteins, NK cells were fixed with Phosflow Lyse/Fix buffer, followed by permeabilization with Phosflow Perm buffer III (BD) and staining with antibodies to S6 phosphorylated at Serc235 and Serc236 (D57.2.2E; Cell Signaling Technology), Akt phosphorylated at Serc473 (M89-61; BD), and Thrc308 (J1-223.371; BD). Flow cytometry data were acquired on LSRII or LSR Fortessa (BD) and analyzed using FlowJo software (Tree Star). Net mean fluorescence intensity (ΔMFI) was calculated. Expression levels were presented as fold relative to unstimulated, set as 1.
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