Ix permeabilization buffer

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom

The IX permeabilization buffer is a solution used to create temporary pores or openings in the cell membrane, allowing the introduction of molecules into the cell interior. The buffer facilitates the entry of various substances, such as antibodies, dyes, or nucleic acids, for analysis or manipulation purposes. The core function of the IX permeabilization buffer is to temporarily permeabilize the cell membrane without compromising the overall integrity of the cell.

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Permeabilization buffer (703 citations)

2 protocols using ix permeabilization buffer

Comprehensive flow cytometry protocol

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FACS staining was external unless otherwise specified. Cells were stained for LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm Excitation (Invitrogen, UK). Internal staining: cells were fixed with 2% formaldehyde (Sigma Aldrich, UK) and permeabilized with IX permeabilization buffer (eBioscience, UK). The following antibodies were used for flow cytometric analysis: anti-CD4-PerCp-Cy5.5 (RPA-T4), anti-CD19-PB (HIB19), anti-CD38-PerCP-Cy5.5 (HIT2), anti-IgD-FITC (IA6-2), anti-CXCR5-PB (J252D4), anti-CD27-PE-Cy7 (M-T271), anti-CD34-FITC (561), anti-CD68-APC (Y1/82A), anti-CD83-BV421 (HB15e), anti-ICOS-APC (C398.4A) (Biolegend, UK), anti-CD20-PO (HI47, Life technologies, UK), anti-CD138-APC (B-A38, Beckman Coulter, UK), anti-CD10-PE-Cy7 (HI10a), anti-CD45-RA-FITC (T6D11), anti-CD161-PE (191B8), anti-IgM-APC (PJ2-22H3), anti-CXCR4-APC (12G5, Milteny Biotec, UK), anti-CD3-FITC (ICHT1, R&D Systems, UK), anti-LLT1-PE (402659 R&D), anti-IgG isotype control-PE, IgG1 and IG2A isotype controls (R&D Systems, UK), anti-mouse IgG-PE (R&D Systems, UK).
Novel mouse anti-human CLEC2D (LLT1) monoclonal antibodies were generated, clones 359.7G7, mIgG1 and 359.2H7, mIgG2al. All purified antibodies are dialyzed against PBS, are low in endotoxin (< 2EU/mg), and are filtered sterile.

Llibre A., López-Macías C., Marafioti T., Mehta H., Partridge A., Kanzig C., Rivellese F., Galson J.D., Walker L.J., Milne P., Phillips R.E., Kelly D.F., Freeman G.J., El Shikh M.E., Klenerman P, & Willberg C.B. (2016). LLT1 and CD161 Expression in Human Germinal Centers Promotes B Cell Activation and CXCR4 Downregulation. The Journal of Immunology Author Choice, 196(5), 2085-2094.

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Multicolor Flow Cytometry Immunophenotyping

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For external staining, cells from cell lines or PBMCs in PBS were incubated with anti-surface antibodies at room temperature (RT) for 20 min. Live/dead staining was performed using LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit(Invitrogen), at 633 or 635 nm excitation.
For internal staining, cells were fixed with 2% formaldehyde (Sigma Aldrich) in PBS for 10 min and permeabilized with IX permeabilization buffer (eBioscience) in water.
The following antibodies were used: CD3-FITC (BioLegend, Catalog No. 300406, clone UCHT1, Mouse IgG1, k), CD8-PerCP-Cy5.5 (BioLegend, Catalog No. 344710, clone SK1, Mouse IgG1, k), CD38-PerCP-Cy5.5 (BioLegend, Catalog No. 303522, clone HIT2, Mouse IgG1, k), CD56-APC (Biolegend, Catalog No. 318310, clone HCD56, Mouse IgG1, k); CD19-BV421 (BD Bioscience, Catalog No. 562441, clone HIB19, mouse IgG1, k); CD4-VioGreen (Miltenyi Biotec, Catalog No. 130-106-712, clone M-T466, Mouse IgG1, k), CD161-PE (Miltenyi Biotec, Catalog No. 130-092-677, clone 191B8, Mouse IgG2a), IgG2A isotype control (R&D Systems, Catalog No. MAB003, mouse); and 2H7 mAb. When non-conjugated primary antibodies were used, a secondary rat anti-mouse IgG2A-PE (R&D Systems, Catalog No. F0129, clone 344701, IgG1) was used.
FACS analysis was performed on Miltenyi Biotec MACSQuant cytometer and analyzed with FlowJo Version 9.6.2 software (TreeStar).

Llibre A., Garner L., Partridge A., Freeman G.J., Klenerman P, & Willberg C.B. (2016). Expression of lectin-like transcript-1 in human tissues. F1000Research, 5, 2929.

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