Intracellular staining permeabilization wash buffer 10x

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Intracellular Staining Permeabilization Wash Buffer (10X) is a 10X concentrated buffer solution designed for the permeabilization and washing of cells during intracellular staining procedures. The buffer facilitates the access of antibodies to intracellular antigens, allowing for the detection and analysis of intracellular proteins.

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Permeabilization Wash Buffer Intracellular staining buffer Wash buffer Permeabilization buffer Staining buffer

4 protocols using intracellular staining permeabilization wash buffer 10x

Comprehensive Immunological Profiling of Tumor Cell Lines

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4T1, H22, HepG2, NIH‐3T3, and Panc02 were purchased from BeNa Culture Collection, China. Anti‐mouse PD‐1 antibody (PD‐1 Ab, BE0146‐100 mg) was purchased from Bio X Cell. Anti‐human PD‐1 Ab was obtained as a gift from Livzon Pharmaceutical Group Inc., China. Flow cytometry antibodies included PE anti‐mouse CD274 (B7‐H1, PD‐L1) (124308), APC/Cyanine7 anti‐mouse CD45 (103132), FITC anti‐mouse CD3 (100204), Brilliant Violet 421 anti‐mouse CD4 (100563), PE/Cy7 anti‐mouse CD8a (100722), PE anti‐mouse IFN‐γ (505808), Alexa Fluor 647 anti‐human/mouse Granzyme B (515406), APC anti‐mouse Ki‐67 (652406), PE anti‐mouse/human CD44 (103008), PE anti‐mouse Ly‐6G/Ly‐6C (Gr‐1) (108407), APC/Cyanine7 anti‐mouse/human CD11b (101226), Brilliant Violet 421 anti‐mouse F4/80 (123137), PE anti‐mouse CD86 (105008), PE/Cy7 anti‐mouse CD206 (MMR) (141720), Alexa Fluor 647 anti‐mouse FOXP3 (126408), APC anti‐mouse CD49b (pan‐NK cells) (108910), PE anti‐mouse CD25 (102008), PE anti‐mouse/human CD44 (103008), APC anti‐mouse CD62L (104412), Zombie Aqua Fixable Viability Kit (423102), Cell Activation Cocktail (with Brefeldin A) (423304), True‐Nuclear Transcription Factor Buffer Set (424401), and Intracellular Staining Permeabilization Wash Buffer (10X) (421002) were purchased from BioLegend.

Liu X., Ye N., Liu S., Guan J., Deng Q., Zhang Z., Xiao C., Ding Z., Zhang B., Chen X., Li Z, & Yang X. (2021). Hyperbaric Oxygen Boosts PD‐1 Antibody Delivery and T Cell Infiltration for Augmented Immune Responses Against Solid Tumors. Advanced Science, 8(15), 2100233.

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Tumor Digestion and Single-Cell Analysis

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Tumors were digested into single-cell suspensions as follows: Tumors were cut into small pieces using a scalpel and needle, and incubated in 40 mL digest media comprising RPMI-1640 with 2 mg/mL collagenase A (Roche, Cat. No. 11088793001) and 40units/mL DNase-I (Sigma-Aldrich, Cat. No. D5025) at 37°C with agitation for 1 hour. Tumor digests were then filtered through a 70 μm cell strainer (BD Falcon) and resuspended in FACS buffer (PBS + 2% FBS + 1 mM EDTA) for staining. Spleens were mashed through a 70 μm cell strainer and resuspended in FACS buffer for staining. Antibodies included CD3 (clone 145–2 C11), CD4 (RM4-R), CD8 (53–6.7), CD44 (IM7), CD62L (MEL-14), TIM-3 (B8.2 C12), LAG-3 (C9B7W), PD-1 (29 F.1A12), IFN-γ (XMG1.2), and TNF (MP6-XT22), all from BioLegend (San Diego, CA). Samples were acquired on an Accuri C6 Flow Cytometer (BD Biosciences) and analyzed with the FlowJo software (version 10.4). For cytokine assessment, digested cells were plated at 10⁶ cells/mL in a 96 well round bottom plate with Cell Activation Cocktail (BioLegend) overnight prior to intracellular staining, as recommended by the manufacturer using Fixation Buffer (BioLegend) and Intracellular Staining Permeabilization Wash Buffer 10X (BioLegend). Gating strategy is shown in Supplementary Figure S6.

White M.G., Szczepaniak Sloane R., Witt R.G., Reuben A., Gaudreau P.O., Andrews M.C., Feng N., Johnson S., Class C.A., Bristow C., Wani K., Hudgens C., Nezi L., Manzo T., De Macedo M.P., Hu J., Davis R., Jiang H., Prieto P., Burton E., Hwu P., Tawbi H., Gershenwald J., Lazar A.J., Tetzlaff M.T., Overwijk W., Woodman S.E., Cooper Z.A., Marszalek J.R., Davies M.A., Heffernan T.P, & Wargo J.A. (2021). Short-term treatment with multi-drug regimens combining BRAF/MEK-targeted therapy and immunotherapy results in durable responses in Braf-mutated melanoma. Oncoimmunology, 10(1), 1992880.

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Comprehensive Immune Cell Analysis

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Fluoroisothiocyanate, PE/dazzle, allophycocyanin, and phycoerythrin-labeled CD3, CD4, CD8, CXCR4, CXCR7, CD45R, HLA-DR, GATA3, Ki-67, Helios, and FOXP3 human monoclonal antibodies, RBC lysis buffer (10X), intracellular staining permeabilization wash buffer (10X), and fixation buffer were purchased from BioLegend (San Diego, USA). GolgiStop was purchased from BD Biosciences (San Diego, USA). RPMI 1640 medium, phorbol myristate acetate (PMA), and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). TRIzol was purchased from Life Technologies (Paisley, UK). SYBR green and cDNA kits were purchased from Applied Biosystems (Foster City, CA, USA). Primers were synthesized from GenScript (Piscataway, NJ, USA).

Alhosaini K., Ansari M.A., Nadeem A., Attia S.M., Bakheet S.A., Al-Ayadhi L.Y., Mahmood H.M., Al-Mazroua H.A, & Ahmad S.F. (2021). Dysregulation of Ki-67 Expression in T Cells of Children with Autism Spectrum Disorder. Children, 8(2), 116.

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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-2D^b and H-2K^b 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.

Nagaoka K., Sun C., Kobayashi Y., Kanaseki T., Tokita S., Komatsu T., Maejima K., Futami J., Nomura S., Udaka K., Nakagawa H., Torigoe T, & Kakimi K. (2021). Identification of Neoantigens in Two Murine Gastric Cancer Cell Lines Leading to the Neoantigen-Based Immunotherapy. Cancers, 14(1), 106.

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