Intracellular staining buffer set

Manufactured by Thermo Fisher Scientific

Sourced in United States, France

The Intracellular Staining Buffer Set is a laboratory product designed for the detection and analysis of intracellular proteins. The set includes a fixation buffer and a permeabilization buffer, which work together to prepare cells for intracellular staining and flow cytometry analysis.

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10 protocols using intracellular staining buffer set

Immune Cell Profiling in Tumor Tissues

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Anesthetized mice were transcardially perfused with 0.9% NaCl and the tumor mass tissues were identified and dissociated with the Neural Tissue Dissociation Kit per the manufacturer’s instruction (Miltenyi Biotech, Gladbach, Germany). Myelin was removed as per protocol published elsewhere^{29 (link)}. Cells were dissolved in Hank’s Balanced Salt Solution containing FBS. For profiling of microglia, infiltrated macrophage, and myeloid-derived immune cells (TAMs), cells were stained with anti-mouse APC-CD11b, BV510-CD45, PE-Ym1, and eFluor 450-CD16/32 to assess TAM infiltration. For T cell profiling, cells were stained with PE-Cy5 CD8a, APC/Cy7-CD4, PE-FoxP3, APC-CD25, and APC-IFNγ. Intracellular staining of FoxP3 was done using intracellular staining buffer set (eBioscience) according to the manufacturer’s instruction. For detecting immune checkpoint blocker expression, cells were stained with PE-Cy5 CD8a, APC/Cy7-CD4, PE-PD-1, and PE-Cy7-CTLA-4. Samples were acquired with a BD LSRII instrument and analyzed with the Flow Jo (Tree Star) software.

Guan X., Hasan M.N., Begum G., Kohanbash G., Carney K.E., Pigott V.M., Persson A.I., Castro M.G., Jia W, & Sun D. (2018). Blockade of Na/H exchanger stimulates glioma tumor immunogenicity and enhances combinatorial TMZ and anti-PD-1 therapy. Cell Death & Disease, 9(10), 1010.

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Multiparameter Flow Cytometry of Immune Cells

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Splenocyte cell suspensions were resuspended in PBS blocking buffer containing 1% BSA and 5% FCS (Sigma-Aldrich) and incubated for 15 min at 4°C with Fc block CD16/CD32 antibodies (BD Biosciences; 5 μg/mL) to prevent non-antigen-specific binding. Cells (5 × 10⁵) were subsequently stained with antibodies (eBioscience, The Netherlands, unless otherwise stated) against CD69-APC, CD4-PerCP Cy5.5, CXCR3-PE, T1ST2-FITC, RORγ-APC, CD25-Alexa Fluor® 488, FoxP3-PE Cy7, CD196-PE, and Fixable Viability Dye-eFluor® 780 (eBioscience, USA) or matching isotype controls for 30 min at 4°C. Cells were fixed using fixation buffer (eBioscience) or permeabilized for intracellular staining using the intracellular staining buffer set (eBioscience) according to the manufacturer's protocol. Flow cytometry was performed using FACS Canto II (BD Biosciences), and results were analyzed using Flowjo Software V. 10.6.2 (Becton Dickinson & Company (BD). We used fluorescence minus one (FMO) to differentiate between negative and positive staining cell populations (3 (link), 22 (link)).

Ghiamati Yazdi F., Zakeri A., van Ark I., Leusink-Muis T., Braber S., Soleimanian-Zad S, & Folkerts G. (2020). Crude Turmeric Extract Improves the Suppressive Effects of Lactobacillus rhamnosus GG on Allergic Inflammation in a Murine Model of House Dust Mite-Induced Asthma. Frontiers in Immunology, 11, 1092.

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Thymocytes TCR Activation Assay

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Thymocytes isolated from miR-181a^+/− and miR-181a^−/− mice were left untreated or were stimulated for 3 h at 37 °C in the presence of plate-bound αCD3 (17A2, 2.5 μg/ml) and soluble αCD28 (37.51, 2.5 μg/ml). Next, cells were stained for surface CD4 and CD8α and intracellular Nur77 with intracellular staining buffer set (eBioscience).

Łyszkiewicz M., Winter S.J., Witzlau K., Föhse L., Brownlie R., Puchałka J., Verheyden N.A., Kunze-Schumacher H., Imelmann E., Blume J., Raha S., Sekiya T., Yoshimura A., Frueh J.T., Ullrich E., Huehn J., Weiss S., Gutierrez M.G., Prinz I., Zamoyska R., Ziętara N, & Krueger A. (2019). miR-181a/b-1 controls thymic selection of Treg cells and tunes their suppressive capacity. PLoS Biology, 17(3), e2006716.

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Multiparameter Flow Cytometry Analysis of PBMCs

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After 24 h of IEC/PBMC co-culture, PBMCs were collected and stained for analysis with flow cytometry. Viability of the cells was determined using Fixable Viability Dye 780-APC Cyanine 7 (eBioscience, Saint Louis, MO, USA). Immunophenotyping and intracellular cytokine staining was performed using antibodies with appropriate isotypes (eBioscience, Saint Louis, MO, USA; Invitrogen, Saint Louis, MO, USA) (for the list of antibodies, clones and dilutions, see Table S1). Nonspecific binding was prevented by blocking for 15 min with PBS containing 2.5% FCS and Human FC Block (BD Biosciences, Franklin Lakes, NJ, USA) before extracellular and intracellular staining. Cells were fixated and permeabilized with the FoxP3/Transcription Factor Staining Buffer Set or Intracellular Staining Buffer Set (eBioscience, Saint Louis, MO, USA). PBMC measurements was performed using BD FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA), and data were analyzed by Flowlogic software, version 8.4 (Inivai Technologies, Melbourne, Australia).

Zuurveld M., Ayechu-Muruzabal V., Folkerts G., Garssen J., van‘t Land B, & Willemsen L.E. (2023). Specific Human Milk Oligosaccharides Differentially Promote Th1 and Regulatory Responses in a CpG-Activated Epithelial/Immune Cell Coculture. Biomolecules, 13(2), 263.

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Intracellular Galectin-9 and Cytokine Quantification

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For evaluation of intracellular expression of Galectin-9, cells were stained using a fixable viability dye (Fixable Viability Stain 780, BD Biosciences, France) and anti-CD45 BV510 (BD Biosciences) before fixation, permeabilization and staining using an Intracellular staining buffer set (Thermo Fisher Scientific) according to the manufacturer’s instructions. Intracellular staining was performed for anti-Galectin-9 FITC (Miltenyi Biotec). Cells were analyzed using a Attune analyzer (Thermo Fisher Scientific).
Human CXCL10, CCL5, and TNF were quantified in culture supernatants using multiplex immunoassay (BD Cytometric Bead Array, BD Biosciences). Data were acquired on Attune analyzer (Thermo Fisher Scientific) and analyzed with FlowJo software (BD Biosciences) using dedicated plugin.

Lamarthée B., Callemeyn J., Van Herck Y., Antoranz A., Anglicheau D., Boada P., Becker J.U., Debyser T., De Smet F., De Vusser K., Eloudzeri M., Franken A., Gwinner W., Koshy P., Kuypers D., Lambrechts D., Marquet P., Mathias V., Rabant M., Sarwal M.M., Senev A., Sigdel T.K., Sprangers B., Thaunat O., Tinel C., Van Brussel T., Van Craenenbroeck A., Van Loon E., Vaulet T., Bosisio F, & Naesens M. (2023). Transcriptional and spatial profiling of the kidney allograft unravels a central role for FcyRIII+ innate immune cells in rejection. Nature Communications, 14, 4359.

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Quantifying Islet Cell Populations

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Accuri C6 flow cytometer (BD Biosciences, Heidelberg, Germany) was used to determine the number of insulin-, glucagon-, and somatostatin-positive cells in NPICCs and REPIs. Single cells were prepared by digesting cell clusters with TrypLE solution (Thermo Fisher Scientific), washed with PBS +10% fetal calf serum (FCS), and filtered through a 30 µm pre-separation filter (Miltenyi, Bergisch-Gladbach, Germany). Then, cells were fixed/permeabilized with an intracellular staining buffer set (Thermo Fisher Scientific) and incubated with Fc-Block (anti-mouse CD16/CD32) for 10 min at room temperature. Thereafter, cells were stained with fluorochrome-labeled antibodies against insulin (anti-insulin-AF647, clone T56-706), glucagon (anti-glucagon-PE, clone U16-850), and somatostatin (anti-somatostatin-AF488, clone U24-354) (BD Biosciences). All antibodies were pretested for appropriate dilution and specificity using isotype control antibodies. Antibodies were incubated at 4°C for 30 min, washed two times with permeabilization buffer and analyzed on a flow cytometer with FlowJo software version 10.4 (TreeStar, Ashland, United States).

Honarpisheh M., Lei Y., Zhang Y., Pehl M., Kemter E., Kraetzl M., Lange A., Wolf E., Wolf-van Buerck L, & Seissler J. (2022). Formation of Re-Aggregated Neonatal Porcine Islet Clusters Improves In Vitro Function and Transplantation Outcome. Transplant International, 35, 10697.

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Comprehensive Immune Cell Profiling

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Cell surface markers were first stained, and the cells were then fixed, permeabilized with an intracellular staining buffer set (Thermo Fisher Scientific) following the manufacturer’s protocol, and stained with intracellular or intranuclear markers. As shown in table S7, antibodies were purchased from BD Biosciences (San Jose, CA, USA), eBioscience (Thermo Fisher Scientific), or BioLegend (San Jose, CA, USA). T_regs were defined as CD4⁺CD25⁺FOXP3⁺, T_FH cells as CD4⁺CD44⁺CXCR5⁺PD-1⁺Bcl6⁺, T_FR cells as CD4⁺CD44⁺CXCR5⁺PD-1⁺Foxp3⁺, and GCB cells as B220⁺CD4-CD95⁺GL-7⁺. See fig. S11 for the detailed gating strategies. Flow cytometry was performed using FACSAria II (BD Biosciences), and the data were analyzed using FlowJo v10.0.7 software (Tree Star Inc., Ashland, OR, USA).

He J., Chu Y., Li J., Meng Q., Liu Y., Jin J., Wang Y., Wang J., Huang B., Shi L., Shi X., Tian J., Zhufeng Y., Feng R., Xiao W., Gan Y., Guo J., Shao C., Su Y., Hu F., Sun X., Yu J., Kang Y, & Li Z. (2022). Intestinal butyrate-metabolizing species contribute to autoantibody production and bone erosion in rheumatoid arthritis. Science Advances, 8(6), eabm1511.

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Multiparametric Immune Profiling of Murine Colon

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Flow cytometry analysis follows a scheme showed in
Fig. S9. Cell
surface markers were first stained, and the cells were then fixed and
permeabilized with an intracellular staining buffer set (Thermo Fisher
Scientific) following the manufacturer's protocol and stained with
intracellular or intranuclear markers. Antibodies (Table S6) were purchased from eBiosciences
(Thermo Fisher Scientific). Memory CD4 cells were defined as CD4 + CD44+,
naïve CD4 cells as CD4 + CD62L+, effective CD8 cells as
CD8 + CD38 + H-2 kb+, γδT cells as CD3 + TCR gamma/delta + . Flow cytometry
was performed using FACSAriaTMII (BD Biosciences) and the data was analyzed
using FlowJo v10.0.7 software (Tree Star Inc., Ashland, OR, USA).
The colon tissues were embedded in paraffin, sectioned and
stained with hematoxylin and eosin (H&E). A microscopic assessment of
lymphatic follicles was performed, as well as immunohistochemistry assay of
CD4+, CD8+, and Treg (CD4 + CD25 + FOXP3 + ) cells with corresponding
antibodies (Table
S6).

Lyu W., Meng Q., Xiao J., Li J., Wang J., Qiu Z., Song X., Zhu H., Shao C., Chu Y., Zhou Q., Li T., Jean-Pierre R., Yu J., Han Y, & Kang Y. (2021). Gut lactate-producing bacteria promote CD4 T cell recovery on Anti-retroviral therapy in HIV-infected patients. Computational and Structural Biotechnology Journal, 19, 2928-2937.

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Multiparametric Flow Cytometry Analysis

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For surface staining, immune cells in skin tissues were first blocked with anti-mouse CD16/32 (101302, Biolegend) for 30 min at 4°C in PBS containing 1% FBS. After washing, cells were stained with following antibodies: anti-mouse TCRβ-AF700(109224, Biolegend), anti-mouse TCR γ/δ-FITC antibody (118106, Biolegend), anti-mouse CD8α-PE (100708, Biolegend), anti-mouse CD11c-PE (117308, Biolegend), anti-mouse F4/80-AF488 (123120, Biolegend), anti-mouse NK1.1-APC(108710, Biolegend), anti-mouse B220-Alexa Fluor 700 (103232, Biolegend), anit-mouse Ly6C (128026, Biolegend), anti-mouse I-A/I-E-BV605 (107639, Biolegend), anti-mouse CD3ε-BUV737 (612771, BD Bioscience), anti-mouse CD11b-BUV737 (612800, BD Bioscience; For intracellular staining, cells were washed with PBS, fixed and permeabilized with Intracellular Staining Buffer Set (00-5523-00, Invitrogen) according to the manufacturer’s protocol. Cells were stained for 1 h at 4°C with different antibodies: anti-mouse TNFα-BV605 (506329, Biolegend), anti-mouse IL-17A-Pacific blue (506918, Biolegend), anti-mouse IL-36α-APC (32103-05161, Assaypro), anti-mouse IL-36γ (PAL621Mu01, Cloud-clone Corp), or control antibody (goat anti-rabbit IgG, 111-136-144, Jackson ImmunoResearch). Cells were acquired either by BD FACS Fortessa or BD FACS Aria II Cell Sorter. All the data were analyzed using FlowJo 10.1.

Tamaru Y, & Doi R.H. (1999). Three Surface Layer Homology Domains at the N Terminus of the Clostridium cellulovorans Major Cellulosomal Subunit EngE. Journal of Bacteriology, 181(10), 3270-3276.

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Multiparametric Flow Cytometry Analysis

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Hao J., Jin R., Zeng J., Hua Y., Yorek M.S., Liu L., Mandal A., Li J., Zheng H., Sun Y., Yi Y., Yin D., Zheng Q., Li X., Ng C.K., Rouchka E.C., Egilmez N.K., Jabbari A, & Li B. (2022). Consumption of fish oil high-fat diet induces murine hair loss via epidermal fatty acid binding protein in skin macrophages. Cell reports, 41(11), 111804.

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