Peripheral blood, spleen, and tumour tissue from mice were collected and processed into single-cell suspensions. Staining was carried out as per the antibody usage instructions (detailed antibody information was provided in the Additional file 8: Table S1 ). Nuclear expression staining was fixed and permeabilised using the True-Nuclear TM Transcription Factor Buffer Set (424,401, Biolegend, USA). Cytokine staining in the cytoplasm was treated with Wash Buffer (421,002, Biolegend), followed by fixation with intracellular staining fixation buffer (420,801, Biolegend) and permeabilisation. Antibody staining was performed at room temperature in the dark for 30 min, and the cells were washed and resuspended in PBS. Data were acquired using flow cytometry (BD FACSCanto II, USA) and analysed with FlowJo 10.8.1 software.
Intracellular staining fixation buffer
Manufactured by BioLegend
Intracellular Staining Fixation Buffer is a laboratory reagent used to fix and permeabilize cells, allowing for the detection of intracellular proteins and antigens. It maintains the structural integrity of cells while enabling the penetration of antibodies or other detection probes into the cell interior.
Automatically generated - may contain errors
Lab products found in correlation
Wash buffer, by BioLegend (1 mentions)
True nuclear transcription factor buffer set, by BioLegend (1 mentions)
Live dead fixable violet, by Thermo Fisher Scientific (1 mentions)
Versacomp antibody capture kit, by Beckman Coulter (1 mentions)
Anti ifn γ pe, by BioLegend (1 mentions)
Cell activation cocktail with brefeldin a, by BioLegend (1 mentions)
Permeabilization wash buffer, by BioLegend (1 mentions)
Collagenase, by Fujifilm (1 mentions)
Zombie yellow fixable viability kit, by BioLegend (1 mentions)
Intracellular staining permeabilization wash buffer 10x, by BioLegend (1 mentions)
Fixable viability dye efluor 450, by Thermo Fisher Scientific (1 mentions)
Anti cd16 32 mab 2.4g2, by BioXCell (1 mentions)
Apc conjugated anti ifn γ mab, by BioLegend (1 mentions)
Rpmi 1640, by Nacalai Tesque (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Intracellular staining permeabilization wash buffer, by BioLegend (1 mentions)
T bet 4b10, by BioLegend (1 mentions)
Irf4 3e4, by Thermo Fisher Scientific (1 mentions)
Live dead aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using intracellular staining fixation buffer
1
Single-Cell Immune Profiling of Tumor Microenvironment
2
Multiparametric Phenotyping of DRG Cells
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DRG cells from naïve, day 7, and day 14 PTX-treated female and male WT and cHET mice were incubated at RT for 30 min with Live/Dead Fixable Violet (Thermo). Cells were pre-incubated with CD16/32 for 10 min prior to the addition of antibodies or isotype (S5 Table in S1 File ) for 20 min at 4°C. Cells were washed with FACs buffer (1× PBS, 1% FBS) and fixed at RT for 20 min with Intracellular Staining Fixation Buffer (BioLegend). Cells were washed with 2× Cyto-FastTM Perm Wash solution (CFPWS; BioLegend) and incubated with PGP9.5 for 20 min at RT. Cells were washed with 2× CFPWS and incubated with donkey anti-rabbit Cy3 for 20 min at RT. VersaComp Antibody Capture Kit (Beckman Coulter) was used to set compensation to correct for spectral overlap. Unstained DRG cells were used to check for auto-fluorescence. Beckman Coulter Cyto-FLEX S System B2-R3-V4-Y4 was used to acquire >100,000 events in the live (Aqua+) DRG cells. Data was analyzed with FlowJoTM 10.8.2.
3
Activation and Cytotoxicity of CD8+ T Cells
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CD8+ T cells were isolated from the spleens of 6‐week‐old healthy C57BL/6 female mice using the CD8+ T cell MACS negative selection kit (Biolegend, San Diego, USA) according to the manufacturer's instructions, and then cultured in complete RPMI 1640 medium supplemented with 20 ng mL−1 IL‐2 for 6 days. The CD8+ T cells were co‐cultured with the above mature BMDCs at the DC/T cell ratio of 1:10 for another 24 h. The cells were stimulated with Cell Activation Cocktail (with Brefeldin A, Biolegend, No 423303) for 2 h. The cells were stained with anti‐mouse CD8‐PE/Cyanine 7 antibody (Biolegend, No 100722, clone 53–6.7), and then fixed with intracellular staining fixation buffer (Biolegend, No 420801) and permeabilized with permeabilization wash buffer (Biolegend, No 421002) according to the manufacturer's instructions. The cells were further stained with anti‐mouse Granzyme‐B‐Alexa Fluor 647 (Biolegend, No 515406, clone GB11) or anti‐IFN‐γ‐PE (Biolegend, cat. No 505808, clone XMG1.2) for 30 min. The cells were washed with PBS and subjected to flow cytometric analysis.
4
Tumor Dissociation and Single-Cell Analysis
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Tumors were cut into pieces and incubated in RPMI-1640 (Nacalai Tesque) supplemented with 1% FBS, 10 mM HEPES, 0.2% collagenase (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and 2 KU/mL DNase I (Sigma-Aldrich) for 40 min at 37 °C. All material was passed through a 70 μm cell strainer to obtain single cell suspensions. After staining dead cells using a Zombie Yellow Fixable Viability Kit (BioLegend) and blocking of Fc receptors with anti-CD16/32 mAb (2.4G2, Bio X Cell), the cells were stained with mAbs for cell surface antigens, H-2Db and H-2Kb dimers. For intracellular cytokine staining, 1 × 105 cells were stimulated with peptides, 1 × 105 YTN2 or 1 × 105 YTN16 cells in the presence of 10 μg/mL brefeldin A (Sigma-Aldrich) for 4 h. After staining dead cells with the Fixable Viability Dye eFluor 450 (Thermo Fisher Scientific) and blocking Fc receptors with anti-CD16/32 mAb, the cells were stained with mAbs for cell surface antigens, followed by fixation, permeabilization, and staining with APC-conjugated anti-IFN-γ mAb (BioLegend) using Intracellular Staining Fixation Buffer and Intracellular Staining Permeabilization Wash Buffer (10X) (BioLegend) according to the manufacturer’s protocols.
5
Multiparametric Analysis of Cellular States
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Cells were labelled with LIVE/DEAD™ Aqua Dead Cell Stain Kit (Invitrogen) and then fixed in Intracellular Staining Fixation Buffer (BioLegend) and permeabilised in Intracellular Staining Permeabilization Wash Buffer (BioLegend). Antibodies against eIF5a (EP527Y, Abcam), hypusine (Hpu24, Creative Biolabs), IFNg (XMG1.2, BioLegend), TNFa (MP6-XT22, eBioscience), IRF4 (3E4, eBioscience), TBET (4B10, BioLegend), CDK1 (A17, Abcam), CD25 (PC61, BioLegend), CDC45 (EPR5759, Abcam) and puromycin (12D10, Millipore) were used; for cell cycle analysis, fixedpermeabilised cells were stained with Hoechst 33342 (Sigma) and Ki-67 antibody (BD Biosciences). Cells were incubated with primary antibodies for 16 hours at 4°C, washed, followed by staining with Goat anti-rabbit Alexa Fluor 647 secondary antibody (Thermo Fisher Scientific) for 1 hour at RT. All analyses were done on singlet live events.
All acquisitions were done on a MACSQuant flow cytometer (Miltenyi Biotec) .
All acquisitions were done on a MACSQuant flow cytometer (Miltenyi Biotec) .
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