Intracellular staining kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Intracellular Staining Kit is a laboratory product designed for the detection and analysis of intracellular proteins or molecules within cells. The kit provides the necessary reagents and protocols for the preparation, fixation, permeabilization, and staining of cells to enable the flow cytometric analysis of intracellular targets.

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Lab products found in correlation

Ionomycin, by Merck Group (24 mentions) Phorbol myristate acetate (pma), by Merck Group (18 mentions) Golgistop, by BD (12 mentions) Brefeldin a, by Thermo Fisher Scientific (10 mentions) Facscanto 2, by BD (8 mentions) Phorbol 12 myristate 13 acetate, by Merck Group (4 mentions) Ionomycin, by Thermo Fisher Scientific (4 mentions) Facsdiva software, by BD (3 mentions) Monensin, by Merck Group (3 mentions) Ionomycin, by Enzo Life Sciences (3 mentions) Monensin, by Enzo Life Sciences (3 mentions) Monensin, by Thermo Fisher Scientific (3 mentions) Il 17, by Thermo Fisher Scientific (3 mentions) Facsdiva, by BD (3 mentions) Leukocyte activation cocktail, by BD (3 mentions) Facscalibur flow cytometer, by BD (3 mentions) Fixable viability dye, by Thermo Fisher Scientific (3 mentions) Foxp3 staining kit, by Thermo Fisher Scientific (2 mentions) Protein transport inhibitor, by Thermo Fisher Scientific (2 mentions) Percp cy5.5 conjugated anti cd4 antibodies, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Foxp3 intracellular staining kit (47 citations) Intracellular staining buffer kit (7 citations) Intracellular cytokine staining kit (4 citations) Intracellular Transcription Factor Staining kit (3 citations) Kits for intracellular staining (2 citations) Intracellular staining fixation & permeabilization kit (2 citations) Annexin V Staining with Surface and Intracellular Staining kit (2 citations) Permeabilization kit for intracellular staining (2 citations) Intracellular transcription factor staining reagent kit (2 citations) Foxp3/Transcription Factor intracellular staining kit (2 citations)

84 protocols using intracellular staining kit

Monocyte-CD4+ T Cell Coculture Assay

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Monocytes or DCs (10,000 cells/200 μl/well) were cocultured with autologous CD4⁺ T cells at a ratio of 1:10 in U-bottom 96-well plates and stimulated with (20 μg/ml) γ-irradiated M. tuberculosis or M. tuberculosis-derived cell wall, cell membrane, or cytoplasmic fractions (10 μg/ml) for 5 days. After 5 days, cell-free supernatants were collected, and T cells were activated with phorbol myristate acetate (50 ng/ml) and ionomycin (500 ng/ml, Sigma-Aldrich, France), along with GolgiStop (BD Biosciences), for 4 h. For the analysis of IL-17⁺CD4⁺ T cells and IL-10⁺CD4⁺ T cells in the cocultures, surface staining was performed with fluorescence-conjugated mAbs to CD4. Then, cells were fixed, permeabilized using intracellular staining kit (eBioscience), and incubated at 4°C with fluorescence-conjugated mAbs to IL-17A, IL-10, pSTAT3, and RORC. Samples were acquired by using LSR II (BD Biosciences) flow cytometry, and data were analyzed by BD FACS DIVA software (BD Biosciences).
For the analysis of FoxP3⁺CD4⁺ T in the cocultures, surface staining was performed with fluorescence-conjugated mAb to CD4. Then, cells were fixed, permeabilized using intracellular staining kit (eBioscience), and incubated at 4°C with fluorescence-conjugated mAb to FoxP3.
For the analysis of PD-1 on CD4⁺ T cells, surface staining was performed with fluorescence-conjugated mAbs to CD4 and PD-1.

Stephen-Victor E., Sharma V.K., Das M., Karnam A., Saha C., Lecerf M., Galeotti C., Kaveri S.V, & Bayry J. (2016). IL-1β, But Not Programed Death-1 and Programed Death Ligand Pathway, Is Critical for the Human Th17 Response to Mycobacterium tuberculosis. Frontiers in Immunology, 7, 465.

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Intracellular Cytokine Staining of CD4+ T Cells

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The CD4⁺ T cells were stimulated with ionomycin (800 ng/mL; Sigma-Aldrich) and phorbol 12-myristate 13-acetate (50 ng/mL; Sigma-Aldrich) for 5 h, and Protein Transport Inhibitor (Invitrogen) was added for the last 2 h. Phycoerythrin (PE)-conjugated anti-IL-17 antibodies (eBioscience, CA, USA) and an intracellular staining kit (Life Technologies) were used. Fluorescein isothiocyanate (FITC)-conjugated anti-Foxp3 antibodies and a Foxp3 staining kit (Invitrogen) were used following the manufacturer’s guidelines. PerCP-Cy5.5-conjugated anti-CD4 antibodies (eBioscience), and PE-conjugated anti-OX40 (BioLegend) antibodies were used for surface staining. The analysis was carried out using a FlowSight system (Amnis, TX, USA).

Alzahrani A, & Hanieh H. (2020). Differential modulation of Ahr and Arid5a: A promising therapeutic strategy for autoimmune encephalomyelitis. Saudi Pharmaceutical Journal : SPJ, 28(12), 1605-1615.

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Intracellular Cytokine Staining of T Cells

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Isolated CD4⁺ T cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 800 ng/mL ionomycin (Sigma-Aldrich) for 5 h, with Protein Transport Inhibitor (Invitrogen) added for the final 2 h. An Intracellular Staining kit (Life Technologies) and phycoerythrin (PE)-conjugated anti-IL-17 antibodies (eBioscience) were used following the manufacturer’s instructions. Foxp3 was stained by using a Foxp3 Staining kit (Invitrogen) including fluorescein isothiocyanate (FITC)-conjugated anti-Foxp3 according to the manufacturer’s instructions. For surface staining, PerCP-Cy5.5-conjugated anti-CD4 antibodies and FITC-conjugated anti-CD45 antibodies from eBioscience were used. The analysis was performed by using a FlowSight system (Amnis).

Abdullah A., Maged M., Hairul-Islam M. I., Osama I. A., Maha H., Manal A, & Hamza H. (2019). Activation of aryl hydrocarbon receptor signaling by a novel agonist ameliorates autoimmune encephalomyelitis. PLoS ONE, 14(4), e0215981.

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Intracellular Cytokine Analysis in Lymph Nodes

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For analysis of intracellular cytokine production, single-cell suspension from pooled peripheral lymph nodes were stimulated for 5 hours with phorbol 12-myristate 13- acetate (PMA, 20 ng/ml) and ionomycin (1g/ml) in the presence of 1mM monensin (all from Sigma, USA). Cells were stained with antibodies for CD4, CD8, IFN? and TNFa using the intra-cellular staining kit (Life Technologies). Recipient CD4 and CD8 gated cells (H-2Kb⁺H-2Kd⁺) were analyzed for intracellular cytokines by flow-cytometry as before [1 (link)]. The serum cytokines (IFNγ, TNFα, IL-4 and IL-10) were analyzed using ELISA-MAX™ kits, using the manufacturer’s instructions (BioLegend).

Venkatadri R., Sabapathy V., Dogan M., Sharma R., Mohammad S., Via C.S, & Sharma R. (2021). Hybrid cytokine IL233 renders protection in murine acute graft vs host disease (aGVHD). Cellular immunology, 364, 104345.

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Flow Cytometry for Immune Cell Phenotyping

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For surface markers, MSCs were collected to stain with relevant fluorochrome-conjugated monoclonal antibodies (mAbs): anti-mouse CD29, CD90, CD44, CD34, CD45, and CD11b from eBioscience. Lymph nodes were isolated from mice and ground in culture medium. The suspensions were filtered through 70-μm cell strainers. For intracellular cytokine staining, single cell suspensions were stimulated with PMA (Sigma-Aldrich, 50 ng/ml), ionomycin (Enzo, 1 μg/ml), and monensin (Enzo, 2 μg/ml). After 5 h, cells were stained with anti-CD4 mAb (eBioscience), fixed, permeabilized, and stained with anti-IFN-γ or IL-17 mAbs (eBioscience) according to the Intracellular Staining Kit (Invitrogen, Carlsbad, CA) instructions. For Treg cells staining, anti-CD4, anti-CD25, and anti-Foxp3 mAbs (eBioscience) were performed following Foxp3 Staining Buffer Set (eBioscience) protocols. Flow cytometry was performed using the BD FACSCanto II (Becton Dickinson) and data were analyzed using FlowJo software (Becton Dickinson) (24 (link)).

Tian J., Zhu Q., Zhang Y., Bian Q., Hong Y., Shen Z., Xu H., Rui K., Yin K, & Wang S. (2020). Olfactory Ecto-Mesenchymal Stem Cell-Derived Exosomes Ameliorate Experimental Colitis via Modulating Th1/Th17 and Treg Cell Responses. Frontiers in Immunology, 11, 598322.

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Th1, CTL, and MDSC Identification

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Resuspended PBMCs (1×10⁶) were cultured in 1 ml 10% fetal bovine serum RPMI 1640 medium in the presence of 50 ng/ml PMA (Sigma-Aldrich, California, USA) and 1 μg/ml of ionomycin (Sigma-Aldrich). After 1 hour, 1 μg/ml brefeldin-A (eBioscience, San Diego, USA) was added to the culture for 4 hours at 37℃ and 5% CO2. Stimulated PBMCs were stained with PE-Cy5-conjugated anti-CD3 mAb and FITC-conjugated anti-CD8 mAb (eBioscience), then fixed, permeabilized and stained with anti-IFN-γ mAb (eBioscience) with an Intracellular Staining Kit (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. Th1 were defined as CD3⁺CD8^-IFN-γ⁺, and CTL were defined as CD3⁺ CD8⁺ IFN-γ⁺.
Resuspended PBMCs (1×10⁶) were stained with APC-conjugated anti-HLA-DR mAb (eBioscience), PE-Cy5-conjugated anti-CD11b mAb (Biolegend), FITC-conjugated anti-CD33 mAb (eBioscience), and PE-conjugated anti-ARG-1 mAb (Biolegend); MDSCs were defined as CD33⁺CD11b⁺HLA-DR^-. All data were acquired with a BD FASCalibur flow cytometer and subsequently analyzed with FlowJo software.

Zhou Q., Tang X., Tian X., Tian J., Zhang Y., Ma J., Xu H, & Wang S. (2018). LncRNA MALAT1 negatively regulates MDSCs in patients with lung cancer. Journal of Cancer, 9(14), 2436-2442.

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Multiparameter Flow Cytometry Analysis

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For surface markers, single-cell suspensions were stained with relevant fluorochrome-conjugated monoclonal antibodies(mAbs): anti-mouse CD40 (HM40-3), CD80 (16-10A1), CD86 (GL1), and MHCII (M5/114.15.2) from eBioscience, anti-mouse CD11b (M1/70), Gr-1 (RB6-8C5), Ly6G (1A8), and Ly6C (HK1.4) from Biolegend, For intracellular staining, cells were stimulated with PMA (Sigma-Aldrich, 50 ng/ml), ionomycin (Enzo, 1 µg/ml), monensin (Enzo, 2 µg/ml). After 5 h, cells were stained with antibodies against surface markers, fixed, permeabilized, and stained with anti-IFN-γ mAb (XMG1.2, eBioscience), anti- IL-17 mAb (eBio17B7, eBioscience), or anti-TGF-β mAb (TW7-16B4, eBioscience) according to the Intracellular Staining Kit (Invitrogen) instructions. Flow cytometry was performed using the BD FACSCanto II (Becton Dickinson) and data were analyzed using FlowJo software (Treestar).

Tian J., Hong Y., Zhu Q., Zhou H., Zhang Y., Shen Z., Guo H., Zhang Y., Ai X., Zhao F., Rui K., Xu H, & Wang S. (2020). Mesenchymal Stem Cell Enhances the Function of MDSCs in Experimental Sjögren Syndrome. Frontiers in Immunology, 11, 604607.

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Intracellular HMGN2 Expression in T Cells

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PHA or T-Ag stimulated PBMCs or purified T cell populations were collected and stained with fluorescence-labeled CD4, CD8, or CD44 surface marker or msIgG-PE/FITC isotypes (Biolegend, USA) control. After washing three times with wash buffer, cells were analyzed for HMGN2 expression by using an intracellular staining kit (Invitrogen, USA). Briefly, a volume of 100 μl fixation buffer was added to fix cells, which were then left at 4°C overnight, followed by washing with permeabilization buffer three times in order to permeabilize cells. All of the samples were divided into two tubes, rabbit-anti-human HMGN2 antibody (1 μg/ml) was added into one tube and the same volume PBS was added into the other as the 2^nd-Ab-FITC control. The samples were incubated at 4°C for 1 hour. The cells were washed three times with permeabilization buffer and then incubated with goat anti-rabbit-IgG-FITC secondary antibody (2^nd-Ab-FITC) at 4°C for 1 hour. Finally, the cells were washed with Flow Cytometry buffer and runed on a Beckman coulter FC500 Flow cytometry. Dates were analyzed by using Submit 5.2 software (Beckman Coulter, USA) after gate lymphocytes group on dot plot graph.

Su L., Hu A., Luo Y., Zhou W., Zhang P, & Feng Y. (2014). HMGN2, a new anti-tumor effector molecule of CD8+ T cells. Molecular Cancer, 13, 178.

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Investigating HOXA1's Impact on Antitumor Immunity

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To investigate whether HOXA1 could directly impair antitumor immune responses, 0.9 × 10⁶ LLC cells were transplanted into C57BL/6 mice. After a palpable tumor was formed, tumor-bearing mice were treated by intratumoural injections of pcDNA3.1-HOXA1 every 2–3 days for 3 weeks. Tumor volume and weight were measured, and single-cell suspensions from the spleen, draining lymph nodes and tumors of tumor-bearing mice were collected for analysis. Cells were stimulated with PMA (Sigma-Aldrich, St. Louis, MO, USA, 50 ng/mL), ionomycin (eBioscience, San Diego, CA, USA, 1 µg/mL), and monensin (eBioscience, 2 µg/mL) for 5 h. After that, cells were stained with anti-CD3 and anti-CD4/CD8 mAbs (eBioscience), fixed, permeabilized and stained with anti-IFNγ mAb (eBioscience) according to the Intracellular Staining Kit (Invitrogen, Carlsbad, CA, USA) instructions. Cells obtained from tumor tissues were stained with anti-CD11b and anti-Gr1 mAbs (BioLegend). All of the experiments were approved by the Animal Use Committee of Jiangsu University in accordance with the International Guiding Principles for Biomedical Research Involving Animals.

Tian X., Ma J., Wang T., Tian J., Zhang Y., Mao L., Xu H, & Wang S. (2018). Long Non-Coding RNA HOXA Transcript Antisense RNA Myeloid-Specific 1–HOXA1 Axis Downregulates the Immunosuppressive Activity of Myeloid-Derived Suppressor Cells in Lung Cancer. Frontiers in Immunology, 9, 473.

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Quantification of Th1 Cells in PBMCs

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Separated PBMCs were resuspended at 1 × 10⁶/ml in RPMI-1640 medium containing 10% fetal bovine serum and stimulated with 50 ng/ml of phorbol myristate acetate (PMA; Sigma-Aldrich, California, USA) and 1 μg/ml of ionomycin (Sigma-Aldrich, California, USA) for 2 hours and then incubated for an additional 4 hours in the presence of 1 μg/ml of brefeldin-A (eBioscience, San Diego, USA) at 37 °C and 5% CO₂. After the incubation, the suspended cells were stained with phycoerythrin-cyanin 5 (PE-Cy5) -conjugated anti-human CD3 mAb and fluorescein isothiocyanate (FITC) -conjugated anti-human CD8 mAb (eBioscience, San Diego, USA) against cell surface antigens for thirty minutes at 4 °C in the dark. Cells were then fixed and permeabilized using an intracellular staining kit (Invitrogen, Carlsbad, USA), followed by incubation for 45 minutes at 4 °C in the dark with PE-conjugated anti-human IFN-γ mAb (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The stained cells were analyzed with Accuri C6 (Becton Dickinson, San Jose, USA). To analyze the proportion of Th1 cells, the population of CD3⁺ CD8^- IFN-γ⁺ cells was defined as Th1 cells.

Peng H., Liu Y., Tian J., Ma J., Tang X., Rui K., Tian X., Mao C., Lu L., Xu H., Jiang P, & Wang S. (2015). The Long Noncoding RNA IFNG-AS1 Promotes T Helper Type 1 Cells Response in Patients with Hashimoto’s Thyroiditis. Scientific Reports, 5, 17702.

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