Intracellular fixation and permeabilization buffer kit

Manufactured by Thermo Fisher Scientific

The Intracellular Fixation and Permeabilization Buffer Kit is a laboratory product designed to prepare cells for intracellular staining and flow cytometry analysis. The kit contains reagents for the fixation and permeabilization of cells, which is a necessary step to enable the detection of intracellular proteins and other cellular components.

Automatically generated - may contain errors

Lab products found in correlation

5 protocols using intracellular fixation and permeabilization buffer kit

Multiparametric Flow Cytometry Immunophenotyping

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell suspensions were stained with combinations of following monoclonal fluorescently conjugated antibodies: TCRβ eFluor^® 450 (H57-597, eBioscience, San Diego, CA, USA), CD4 FITC (GK1.5, eBioscience), CD8a PerCP-Cyanine5.5 (53–6.7, eBioscience), CD25 APC (PC61.5, eBioscience), FoxP3 PE (NRRF-30, eBioscience), CD45R/B220 APC-Cyanine7 (RA3-6B2, Biolegend, San Diego, CA, USA), IgD Brilliant violet 650™ (11-26c.2a, Biolegend), CD11b Alexa Fluor^® 700 (M1/70, Biolegend), F4/80 PE (BM8, Biolegend), PD-1 PE-eFluor™ 610 (J43, eBioscience), CTLA-4 PerCP-Cyanine5.5 (UC10-4B9, Biolegend), IL-10 PerCP-Cyanine5.5 (JES5-16E3, Biolegend), LAP PerCP-Cyanine5.5 (TWT-16B4, Biolegend), CD14 FITC (Sa2-8, eBioscience), CD206 APC (MR6F3, eBioscience), MHC-II e Fluor^® 450 (M5/114.15.2, eBioscience). To stain for intracellular murine antigens, cells were first stained for surface antigens, then fixed and permeabilized with intracellular fixation and permeabilization buffer kit (eBioscience) according to the manufacturer’s recommendation. For intracellular IL-10 staining, cells were incubated with PMA (50 ng/ml) and ionomycin (1000 ng/ml) for 2 h, then incubated with Brefeldin A (10.6 μM) and Monensin (2 μM) for 3 h. Data were acquired using BD Fortessa X20 (BD Bioscences, San Diego, CA, USA) and analyzed with FlowJo 7.6 software.

Qiu D., Chu X., Hua L., Yang Y., Li K., Han Y., Yin J., Zhu M., Mu S., Sun Z., Tong C, & Song Z. (2019). Gpr174-deficient regulatory T cells decrease cytokine storm in septic mice. Cell Death & Disease, 10(3), 233.

+ Open protocol

+ Expand

Intracellular Staining Protocol for Flow Cytometry

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were prepared for flow cytometry as previously described above. The intracellular fixation and permeabilization buffer kit (#88-8824, eBioscience) was used for intracellular (cytoplasmic) staining. After extracellular staining and viability dye stain had been performed, 100 μl of intracellular (IC) fixation buffer were added for 1 hr at RT. Following this, the IC buffer was washed twice with permeabilization buffer. The intracellular antibody was diluted in permeabilization buffer for 20–60 min. Cell pellets were resuspended in 100 μl FACS buffer and stored at 4°C until acquisition on the flow cytometer, performed on the same or the following day.

Spear S., Candido J.B., McDermott J.R., Ghirelli C., Maniati E., Beers S.A., Balkwill F.R., Kocher H.M, & Capasso M. (2019). Discrepancies in the Tumor Microenvironment of Spontaneous and Orthotopic Murine Models of Pancreatic Cancer Uncover a New Immunostimulatory Phenotype for B Cells. Frontiers in Immunology, 10, 542.

+ Open protocol

+ Expand

Multiparameter Flow Cytometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell suspensions were obtained from intestines, spleens, and MLNs and stained in PBS 2% FCS with conjugated antibodies to the following markers: TCR-β (eBioscience), TCR-γδ (eBioscience), CD3 (eBioscience), CD45 (eBioscience), CD4 (eBioscience), CD8a (eBioscience), CD62L (eBioscience), CD44 (BD), CD45R (B220; eBioscience), CD357 (GITR; eBioscience), CCR9 (eBioscience), α4β7 (BioLegend), Foxp3 (eBioscience), IgA (eBioscience), IgM (eBioscience), CD25 (eBioscience), CD103 (eBioscience), CD45RB (BioLegend), and Nrp-1 (R&D Systems). Live/dead cell discrimination was obtained using the Aqua Dead Cell Stain kit (Invitrogen). For cytokine production, cells were stimulated for 4 h with 20 ng/ml PMA (Sigma-Aldrich) and 0.5 µg/ml ionomycin (Sigma-Aldrich). Golgi Stop (1,000×; BD) was added during the last 3 h of stimulation. Cells were fixed and permeabilized using intracellular fixation and permeabilization buffer kit (eBioscience) and stained for conjugated anti–IL-17A (eBioscience) and anti–IFN-γ (eBioscience). Flow cytometry data were acquired at FACSCanto II. Data were analyzed with FlowJo software (version 7.6.5).

Rigoni R., Fontana E., Guglielmetti S., Fosso B., D’Erchia A.M., Maina V., Taverniti V., Castiello M.C., Mantero S., Pacchiana G., Musio S., Pedotti R., Selmi C., Mora J.R., Pesole G., Vezzoni P., Poliani P.L., Grassi F., Villa A, & Cassani B. (2016). Intestinal microbiota sustains inflammation and autoimmunity induced by hypomorphic RAG defects. The Journal of Experimental Medicine, 213(3), 355-375.

+ Open protocol

+ Expand

Analyzing T Cell Activation in Tumor Microenvironment

Check if the same lab product or an alternative is used in the 5 most similar protocols

B16-OVA tumor cells infected with scramble sgRNA or sgRAD21 were stained with MHC-I (BioLegend, catalog 116525) and MHC-I SIINFEKL (eBioscience, catalog 17-5743-82). The B16-OVA or ID8-OVA tumor cells were cocultured with T cells (B3Z or OT-I cells) for 24 hours. The LacZ activity and supernatant levels of IL-2 and IFN-γ were examined as previously described (40 (link)). For intracellular cytokine staining, GolgiStop reagent (1,000×; BD Biosciences) was added to the coculture system for 3 hours before staining. First, OT-I cells were stained with fluorescence-labeled antibodies against CD8 (BioLegend, catalog 100706) for 1 hour at 4°C. Next, the cells were fixed and permeabilized using an intracellular fixation and permeabilization buffer kit (eBioscience) and stained with anti–IFN-γ (eBioscience, catalog 17-7311-82) or anti-GZMB (eBioscience, catalog 12-8898-82). The stained cells were then analyzed using flow cytometry.

Deng P., Wang Z., Chen J., Liu S., Yao X., Liu S., Liu L., Yu Z., Huang Y., Xiong Z., Xiao R., Gao J., Liang W., Chen J., Liu H., Hong J.H., Chan J.Y., Guan P., Chen J., Wang Y., Yin J., Li J., Zheng M., Zhang C., Zhou P., Kang T., Teh B.T., Yu Q., Zuo Z., Jiang Q., Liu J., Xiong Y., Xia X, & Tan J. (2022). RAD21 amplification epigenetically suppresses interferon signaling to promote immune evasion in ovarian cancer. The Journal of Clinical Investigation, 132(22), e159628.

+ Open protocol

+ Expand

Immunofluorescence Assay of Connexin 43

Check if the same lab product or an alternative is used in the 5 most similar protocols

For the immunofluorescence assay, both the control and infected cells were seeded on cover slips and incubated at 37°C until 40% to 50% confluence. They were then fixed and permeabilized using an Intracellular Fixation and Permeabilization Buffer kit (eBioscience, San Diego, CA) for 20 minutes at room temperature. The cells were blocked in 10% (w/v) normal goat serum in PBS for 30 minutes at room temperature. Next, the cells were incubated for 2 hours at 37°C with Cx43 antibodies (Cell Signaling Technology), followed by goat anti-rabbit FITC-conjugated IgG, and Rhodamine was used to label the cell nuclei (ZSGB-BIO, China). Digital pictures were taken using a Zeiss LSM 510 Meta Laser scanning confocal microscope.

Lu Y., Wang X.M., Yang P., Han L., Wang Y.Z., Zheng Z.H., Wu F., Zhang W.J, & Zhang L. (2018). Effect of gap junctions on RAW264.7 macrophages infected with H37Rv. Medicine, 97(35), e12125.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!