Flow cytometry permeabilization buffer

Manufactured by R&D Systems

Flow Cytometry Permeabilization Buffer is a solution designed to facilitate the intracellular staining of cells for flow cytometry analysis. The buffer permeabilizes the cell membrane, allowing fluorescent-labeled antibodies or other reagents to access and bind to intracellular targets.

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Lab products found in correlation

Flow cytometry fixation buffer, by R&D Systems (3 mentions) Versene, by Thermo Fisher Scientific (2 mentions) Anti vimentin clone rv202, by BD (2 mentions) Anti fibronectin antibodies clone 10, by BD (2 mentions) Pe conjugated goat anti mouse secondary antibodies, by Thermo Fisher Scientific (2 mentions) Lsrii flow cytometer, by BD (2 mentions) Flow cytometry staining buffer, by R&D Systems (1 mentions) Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions) Facscalibur cytometer, by BD (1 mentions) Roswell park memorial institute (rpmi), by Quality Biological (1 mentions) Percp labeled cd45, by BD (1 mentions) Facscan, by BD (1 mentions) Collagenase a, by Roche (1 mentions) Ack buffer, by Quality Biological (1 mentions)

4 protocols using flow cytometry permeabilization buffer

HCV Core Protein Detection by Flow Cytometry

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Cells were collected by incubation with Accumax™ Cell Detachment Solution (Sigma), pelleted (300xg for 5 minutes), washed twice with Flow cytometry staining Buffer (R&D Systems) and blocked with Human Fc Blocking Solution (R&D Systems) for 15 minutes at 4°C. Cell viability was established with the LIVE/DEAD Fixable Aqua Dead cell stain kit (Life Technologies), following manufacturer instructions.
For HCV core staining, fixation was carried out by using Flow cytometry Fixation Buffer (R&D Systems) and incubating the cells for 10 minutes at room temperature. Permeabilization was performed with Flow cytometry permeabilization Buffer (R&D Systems). After an incubation of 15 minutes at room temperature, cells were stained with 1 μg (per 10⁶ cells) of mouse anti-HCV core monoclonal antibody (Pierce, Thermo Scientific) and Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (Life Technologies) diluted 1/2000. All flow cytometry studies were performed on a FACS Calibur cytometer (BD Biosciences, San Diego, CA, USA) with data analyses conducted using FlowJo 7.6.3 software (Tree Star, Inc., Ashland, OR, USA).

Ulitzky L., Lafer M.M., KuKuruga M.A., Silberstein E., Cehan N, & Taylor D.R. (2016). A New Signaling Pathway for HCV Inhibition by Estrogen: GPR30 Activation Leads to Cleavage of Occludin by MMP-9. PLoS ONE, 11(1), e0145212.

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Multimarker Flow Cytometry Analysis

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Cells were harvested using the non‐enzymatic dissociation buffer Versene (Life Technologies) and collected by centrifugation. Cells were exposed to an anti‐CD324 antibody (E‐cadherin, clone 180224, R & D Systems, Minneapolis, MN). For intracellular staining, cells were exposed to Flow Cytometry Fixation Buffer (R & D Systems) for 20 min at 4 °C in the dark, followed by the addition of Flow Cytometry Permeabilization buffer for 5 min at room temperature (R & D Systems). Cells were exposed to anti‐Vimentin (clone RV202) and anti‐Fibronectin antibodies (clone 10, BD Biosciences, San Jose, CA, used also for western blot) all at optimized antibody dilutions. Primary antibodies were recognized by PE‐conjugated goat anti‐mouse secondary antibodies (Life Technologies) on an LSR II Flow Cytometer (BD Biosciences).

Brozovic A., Duran G.E., Wang Y.C., Francisco E.B, & Sikic B.I. (2015). The miR‐200 family differentially regulates sensitivity to paclitaxel and carboplatin in human ovarian carcinoma OVCAR‐3 and MES‐OV cells. Molecular Oncology, 9(8), 1678-1693.

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Immunophenotyping of Epithelial-Mesenchymal Transition

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Cells were harvested using the non-enzymatic dissociation buffer Versene (Life Technologies) and collected by centrifugation. Cells were exposed to an anti-CD324 antibody (E-cadherin, clone 180224, R & D Systems, Minneapolis, MN). For intracellular staining, cells were exposed to Flow Cytometry Fixation Buffer (R & D Systems) for 20 min at 4 °C in the dark, followed by the addition of Flow Cytometry Permeabilization buffer for 5 min at room temperature (R & D Systems). Cells were exposed to anti-Vimentin (clone RV202) and anti-Fibronectin antibodies (clone 10, BD Biosciences, San Jose, CA, used also for western blot) all at optimized antibody dilutions. Primary antibodies were recognized by PE-conjugated goat anti-mouse secondary antibodies (Life Technologies) on an LSR II Flow Cytometer (BD Biosciences).

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Lung Single Cell Analysis by FACS

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Whole lung single cell suspension was analyzed by FACS analysis with. Briefly mouse lungs were harvested then digested in lung digestion media with 1.0 mg/mL collagenase A (Roche Diagnostics) in RPMI (Quality Biological), and erythrocytes were lysed with ACK buffer (Quality Biological). Cells isolated from the lungs were stained with PerCP-labeled CD45 (BD), APC-Cy7-labeled CD11b (BD), PE-labeled Ly6G (BD), and FITC-Labeled LTA₄H (Bioss). Infiltrating leukocytes were gated from non-leukocytes by the expression of CD45. Next, all CD45^high cells were gated into Ly6G^high and CD11b^high cells (neutrophils) as previously described.(3 (link)) For the purpose of detecting cells containing the LTA₄H in their intra-cellular space, cells were permeabilized with flow cytometry permeabilization buffer (R&D) after stained with CD45 antibody. Cells expressing LTA₄H were separated into two groups, leukocytes (CD45^high) and non-leukocytes (CD45^low). Multi-color detection of the stained cells was performed on a FACScan flow cytometer (BD Biosciences). FlowJo (version 8.8.6.) was used to analyze the data.

Paige M., Wang K., Burdick M., Park S., Cha J., Jeffrey E., Sherman N, & Shim Y.M. (2014). Role of leukotriene A4 hydrolase aminopeptidase in the pathogenesis of emphysema. Journal of immunology (Baltimore, Md. : 1950), 192(11), 5059-5068.

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