Immune population analysis was performed by flow cytometry. The samples were blocked by non-specific staining with TruStain FcX™ PLUS and incubated for 30 min at 4°C in the dark with brilliant violet staining buffer (Biolegend) containing fluorescence-conjugated antibodies (1:100) against the surface markers CD45, CD3, CD4, CD8, NKp46, CD62L, CD44, CD11b, F4/80, and GR-1. The samples stained for intracellular GrB and Ki-67 expression were permeabilized with eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, United States). Finally, the cells were detected using a BD LSRFortessa™ X-20 flow cytometer (BD Biosciences, San Diego, CA, United States).
Trustain fcx plus
Manufactured by BioLegend
Sourced in United States
The TruStain FcX PLUS is a laboratory product designed to block Fc receptors and minimize non-specific binding in flow cytometry experiments. It functions by occupying Fc receptors to prevent unwanted antibody binding, thereby improving the specificity of the staining procedure.
Automatically generated - may contain errors
Lab products found in correlation
Rbc lysis buffer, by BioLegend (5 mentions)
True stain monocyte blocker, by BioLegend (3 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (3 mentions)
Lsrfortessa x 20 flow cytometer, by BD (3 mentions)
Cell staining buffer, by BioLegend (2 mentions)
True nuclear transcription factor buffer set, by BioLegend (2 mentions)
Moflo xdp flow cytometer, by Beckman Coulter (2 mentions)
Cd3 17a2, by BD (2 mentions)
Tumor dissociation kit, by Miltenyi Biotec (2 mentions)
Perm wash, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Cd4 pe, by BioLegend (2 mentions)
Cytoflex s, by Beckman Coulter (2 mentions)
Brilliant violet staining buffer, by BioLegend (1 mentions)
Ebioscience foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Ghost dye red 780 fixable viability dye, by Cell Signaling Technology (1 mentions)
Fitc lineage, by BioLegend (1 mentions)
Sca 1 pe cy7, by BioLegend (1 mentions)
Cd11b fitc, by Thermo Fisher Scientific (1 mentions)
Ultracomp ebeads compensation beads, by Thermo Fisher Scientific (1 mentions)
37 protocols using trustain fcx plus
1
Flow Cytometry Analysis of Immune Populations
2
Multicolor Flow Cytometry Panel for Murine HSCs
3
Flow Cytometric Identification of Hepatic Immune Cells
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Cells were prestained with a 1:100 dilution of Zombie Aqua (Fixable Viability Dye; BioLegend, London, UK) for 20 minutes at 4°C in the dark. After 10 minutes, an equal volume of a 1:100 dilution of TruStain FcX PLUS (anti-mouse CD16/32 antibody) and True-Stain Monocyte Blocker (BioLegend) was added. After a washing step, cells were stained with CD3e-PerCP-Cy5.5, CD19-PerCP-Cy5.5 (eBioscience, Thermo Fisher Scientific), NK1.1-PerCP-Cy5.5, CD103-PerCP-Cy5.5, F4/80-FITC, Ly6G-BV785, Ly6C-BV650 (BioLegend), SiglecF-PerCP-Cy5.5, CD45-APC-Cy7, CD11b-PE-Cy7 and Tim4-PE (BD Biosciences, Erembodegem, Belgium) for 20 minutes at 4°C in the dark. Cells were analyzed with a BD FACSAria Fusion flow cytometer (BD Biosciences) and FlowJo software (FlowJo LLC, BD Biosciences), and gated first as live CD45+ single cells. Subsequently, CD3e+, CD19+, NK1.1+, CD103+ and SiglecF+ cells were eliminated, and CD11b+Ly6C+Ly6G- monocytes, CD11b+Ly6C-F4/80+Tim4+ Kupffer cells (KCs) and CD11b+Ly6C-F4/80+Tim4- monocyte-derived macrophages (MoMfs) were gated.
4
Multicolor Flow Cytometry Immunophenotyping
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Cells were washed with FACS buffer (2% FBS in PBS with 2mM EDTA), followed by a blocking step with anti-mouse Cd16/32 FC block (TruStain FcXPLUS, Biolegend, 156,603,1:200) for 15 min on ice. Cells were then stained with titrated antibodies for Cd45-PE-Cy7 (BD Biosciences, 561,868, clone 30-F11, 1:400), Cd11b-AlexaFluor700 (BD Biosciences, 557,960, clone M1/70, 1:800), Ly6C-PerCP-Cy5.5 (BD Biosciences, 560,525, clone AL-21, 1:200), Ly6G-PacificBlue (Biolegend, 127612, 1:200) in Staining Buffer (Biolegend, 420201) at 4°C for 20min. Cells were washed and resuspended in 300ul of Staining Buffer. Samples were analyzed on BD LSR II cytometer (BD Biosciences). Gating and subpopulation analysis was performed using FlowJo software (BD Biosciences). Reported percentages are given as the percentage of a parental gate, singlets discrimination was based on FSC and SSC scatters.
5
Comprehensive Immune Cell Profiling
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Lymphocytes from splenocytes were treated by red blood cell (RBC) lysis buffer (420302; BioLegend) and debris removal solution kit (Miltenyi Biotec, Bergisch, Gladbach, Germany). Tumor infiltrating lymphocytes (TILs) were treated by RBC lysis buffer and TTDR kit (661563; BD Biosciences). Lymphocytes or monocytes were treated by Fc receptor blocker (TruStain FcX PLUS, S17011E; BioLegend). After discrimination of live/dead cells by zombie aqua (423102; BioLegend) or zombie NIR (423106; BioLegend), cells were stained by PerCP CD3ε (100326; BioLegend), APC/Cy7 CD4 (100525; BioLegend), FITC CD8α (100706; BioLegend), FITC CD11b (101206; BioLegend), PerCP CD11c (117326; BioLegend), BV510 CD27 (124229; BioLegend), PE-Cy7 CD44 (103030; BioLegend), PE CD45 (103106; BioLegend), APC CD62L (104412; BioLegend), PE-Cy7 CD127 (135022; BioLegend), APC CD215(IL-15Rα) (153506; BioLegend), or BV421 KLRG1 (423102; BioLegend). All data were analyzed using FACS lyric or FACS ARIAIII (BD Biosciences) and FlowJo Software ver. 8.1 (BD Biosciences).
6
Hepatic Macrophage Immunophenotyping by FACS
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THP-1 cells and hepatic macrophages in each group were harvested and stained with APC/CY7 anti-mouse CD45 (103116; BioLegend, USA), PE anti-mouse CD11b (101208; BioLegend), APC anti-mouse F480 (123116; BioLegend), PC7 anti-mouse CD206 (141720; BioLegend), PC7 anti-human CD206 (T7-782-T100, EXBIO) and PC5.5 anti-mouse MHC-II (107626; BioLegend) antibodies at 4 °C for 30 min. Hepatic macrophages were detected in the mice by grinding the liver tissues gently, filtering the homogenate through a 80-μm nylon mesh filter, and isolating hepatic mononuclear cells with 40% and 80% Percoll (17-0891-01; GE Healthcare, UK). The isolated cells were stained with the above surface markers after being blocked with anti-mouse CD16/32 (TruStain FcX PLUS; BioLegend) following red blood cell lysis (420301; BioLegend, USA). After staining, the cells were washed three times with PBS and resuspended with 200 μl of 10% BSA diluted with PBS, and analyzed by FACS with a BD FACS Aria II (BD Science, USA). The data were analyzed using FlowJo v10 (TreeStar, Ashland, USA).
7
Comprehensive Immune Cell Analysis of Bronchoalveolar Lavage
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Bronchoalveolar lavage pellets were stained with antibodies and analyzed on a Beckman Coulter “Cytoflex” 13-color cytometer (Beckman Coulter, Brea, CA, United States). Antibodies used include CD16/32 (TruStain FcX PLUS, Biolegend, Catalog 156603), CD45 (APC-Fire 750, Biolegend, Catalog 147714), Ly-6G (Alexa Fluor 488, Biolegend, Catalog 127626), CD11a/CD18 (PE, Biolegend, Catalog 141006), CD11b/CD18 (APC, Biolegend, Catalog 101212), CD62L (BV421, Biolegend, 104435), CD49d (PE, Biolegend 103608, Catalog 103608), CD44 (AAPC, Biolegend, Catalog 103012), and CD162 (BV421, BD Biosciences, Catalog 562807). We identified live/dead cell ratios by using propidium iodide (PI, Fisher Scientific Catalog BDB556463).
8
Quantifying Mitochondrial Transfer in Co-culture
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For assessing mitochondrial transfer rate in the coculture system, 5 × 104 bEnd.3 cells and 5×104 or 15×104 MLO-Y4/MLO-Y4Mito-Dendra2 cells were seeded in 6 well plated. The media were refreshed every 24 hours. After targeted time (24 hours, 48 hours, 72 hours or 7 days), cells were dispatched and stained with Zombie Violet™ Fixable Viability Kit (Biolegend, Cat. 423113), TruStain fcX™ PLUS (Biolegend, Cat. 156603), APC anti-mouse CD31 Antibody (Biolegend, Cat. 102510) sequentially. Cell pellets were then resuspended in 200 µl PBS containing 2% FBS and immediately analyzed with a CytoFLEX Flow Cytometer (Beckman Coulter, California, United States, CytExpert 2.3.1.22). bEnd.3 co-cultured with MLO-Y4 (without Dendra2 fluorescence) were utilized for gating the Dendra2+ bEnd.3 cells. Data were analyzed by FlowJo software version 10.4.
9
Comprehensive Immune Profiling of Tumor Tissues
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Immune population analysis was done by flow cytometry. Tumor tissues were dissociated using a mouse tumor dissociation kit and a gentle MACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Spleens were dissociated by gentle grinding with a syringe plunger. The cells were treated with RBC lysis buffer (Biolegend), suspended in a cell staining buffer (Biolegend), and blocked by nonspecific staining with TruStain FcX™ PLUS. The cells were then incubated for 30 min (4 °C in the dark) with fluorescence-conjugated antibodies (1:100) specific for the lymphocyte markers anti-CD45, anti-CD3, anti-CD8, anti-CD4, and anti-NKp46, and the monocyte markers anti-CD45, anti-CD11b, anti-GR-1, anti-F4/80, anti-MHC Class II, and anti-CD206. Finally, the cells were analyzed using a BD LSRFortessa™ X-20 flow cytometer (BD Biosciences, San Jose, CA, USA) with FlowJo software (BD Biosciences).
10
Tumor-Infiltrating Leukocyte Profiling
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Single-cell suspensions of tumor-infiltrating leukocytes were prepared as described above. Cells were first incubated with 2.5 μg/ml TruStain FcX Plus (BioLegend) in FACS buffer for 10 min on ice. Cells were subsequently stained with fluorochrome-conjugated antibodies (Supplementary Table S1; BioLegend) in FACS buffer for 30 minutes on ice in the dark, followed by a wash with FACS buffer and resuspension in 400 μl of FACS buffer supplemented with 1 μg/ml propidium iodide. In some experiments, the absolute numbers of tumor-infiltrating immune cells were determined using the Precision Count BeadsTM (BioLegend). When staining for FoxP3 in Tregs, cells were first incubated with 1 μl eFluor 450 fixable viability dye (Invitrogen) for 30 minutes on ice in the dark, followed by FcR block and cell surface marker fluorochrome-conjugated antibodies staining. Stained cells were then fixed and permeabilized using BioLegend True Nuclear Transcription Factor Buffer Set and stained with anti-FoxP3 antibody. Samples were analyzed using a LSRFortessa X-20 flow cytometer (BD), and data analysis was performed using FCS Express 7 (DeNovo Software). Gating strategies are shown in Supplementary Figures S1-4.
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