Blood was collected in heparinized tubes from the retro-orbital plexus to assess CD4, CD8, CD14, and TLR-4 surface markers. FITC- or PE-labeled CD4, PE- or FITC-labeled CD8, APC-labeled TLR-4, and PE/Dazzle-labeled CD14 (BioLegend, USA) monoclonal antibodies were used to determine the percentage of CD4+, CD8+, TLR-4+, and CD14+cell surface receptors, respectively. These were added to 100 µL of blood lysed with a lysing buffer (BioLegend, USA). Approximately 20 µL of the fluorescently labeled monoclonal antibodies were added to the suspension of blood leucocytes (CD4 FITC, CD8 PE, CD14 PE/Dazzle-labeled, and TLR-4 APC, BioLegend, USA). These were then incubated at room temperature for 30 min. To stain the intracellular markers IL-17A, NF-κB p65, and TNF-α, the cells were permeabilized, subjected to fixation by standard buffers (BioLegend, USA), and stained with intracellular APC-IL-17A, FITC-NF-κB p65, and PE-TNF-α antibodies (BioLegend, USA; Santa Cruz, Dallas, USA), (Ahmad, Attia, Zoheir, Ashour & Bakheet, 2014 (link)). After centrifugation at 300g for 10 min, the samples were kept at 4 °C until flow cytometry analysis was conducted. Immunostained leukocytes in each sample were acquired using an FC 500 flow cytometer; 10,000 events/sample were then analyzed using CXP software (Beckman Coulter, USA).
Lysing buffer
Manufactured by BioLegend
Sourced in United States
Lysing buffer is a solution used to disrupt cell membranes and release cellular contents, including proteins, nucleic acids, and other intracellular components. It is a core component in various cell biology and molecular biology protocols that require the extraction and analysis of cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Fc500 flow cytometer, by Beckman Coulter (1 mentions)
Cd4 fitc, by BioLegend (1 mentions)
Cd8 pe, by BioLegend (1 mentions)
Tlr4 apc, by BioLegend (1 mentions)
Cxp software, by Beckman Coulter (1 mentions)
Pe labeled cd4, by BioLegend (1 mentions)
Fitc labeled cd8, by BioLegend (1 mentions)
Pe conjugated anti mouse ly 6g, by BioLegend (1 mentions)
Fitc conjugated anti mouse f4 80, by BioLegend (1 mentions)
2 protocols using lysing buffer
1
Flow Cytometric Immunophenotyping of Blood Cells
2
Profiling Immune Cells in Mice by Flow Cytometry
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Flow cytometric analysis was performed as described, with some modifications [26 (link)]. Samples of peripheral blood (0.2 mL) from il17a+/+ and il17a−/− mice were collected from a tail vein in the presence of EDTA as an anticoagulant. The red blood cells (RBS) were removed by incubation with lysing buffer (BioLegend, San Diego, CA, USA) at room temperature for 5 min. The white blood cells (WBC) were collected by centrifugation at 300× g at room temperature for 10 min and washed once with PBS. The Fc receptors were blocked with anti-murine CD16/CD32 at 4 °C for 30 min to prevent nonspecific binding. The cells were then stained with phycoerythrin (PE)-conjugated anti-mouse Ly6G (BioLegend, San Diego, CA, USA) and fluorescein isothiocyanate (FITC)-conjugated anti-mouse F4/80 (BioLegend, San Diego, CA, USA), and subsequently incubated at 4 °C for 60 min in the dark. After staining, the cells were washed with 2 mL of flow cytometric buffer and centrifuged at 300× g at 4 °C for 10 min. The cells were resuspended in 500 μL of flow cytometric buffer, after removing the cell supernatant. All samples were acquired on a cytoflex flow cytometer (Backman, Indianapolis, IN, USA), and data were analyzed using CytExpert software version 2.2 (Backman, Indianapolis, IN, USA).
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