Splenocytes were incubated with TruStain FcX™ PLUS anti-mouse CD16/CD32 (Biolegend; clone: S17011E) for 10 min at 4°C, and then stained with mAbs specific for various cell surface markers. To evaluate the production of IFN-γ and Granzyme B, splenocytes were diluted to 4 × 106 cells/mL and cultured (500 μL/well) in a 24-well plate containing Cell Activation Cocktail (with Brefeldin A; Biolegend) for 6 h. Cells were then harvested and washed twice in cell staining buffer prior to surface marker staining as described above. Cells were then fixed and permeabilized using Intracellular Fixation Buffer & Intracellular Staining Permeabilization Wash Buffer (Biolegend). Intracellular staining was then performed using mAbs specific for IFN-γ and granzyme B, respectively. For Ki-67 staining, freshly isolated splenocytes were stained following the above-mentioned surface and intracellular staining procedures, with no in vitro stimulation. Samples were resuspended in cell staining buffer, tested with BD FACSCelesta flow cytometer, and analyzed using FlowJo software (BD, USA).
Intracellular fixation buffer
Manufactured by BioLegend
Sourced in Switzerland
Intracellular fixation buffer is a solution used for the stabilization and preservation of cellular structures, particularly intracellular proteins and molecules, during the process of sample preparation for flow cytometry or other analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by BioLegend (2 mentions)
Permeabilization buffer, by BioLegend (2 mentions)
Intracellular staining permeabilization wash buffer, by BioLegend (1 mentions)
Facscelesta flow cytometer, by BD (1 mentions)
Cell activation cocktail with brefeldin a, by BioLegend (1 mentions)
Duoset elisa development kit, by R&D Systems (1 mentions)
Anti cd107a bv421, by BioLegend (1 mentions)
Anti cd107a pe cy7, by BioLegend (1 mentions)
Perm wash buffer, by BioLegend (1 mentions)
Facscanto, by BD (1 mentions)
Anti mouse il 10 pe, by Thermo Fisher Scientific (1 mentions)
Pacific blue anti mouse cd4, by BioLegend (1 mentions)
Anti mouse cd3ε pe cy7, by BioLegend (1 mentions)
Anti mouse ifn γ percp cy5, by Thermo Fisher Scientific (1 mentions)
Kaluza software, by Beckman Coulter (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
5 protocols using intracellular fixation buffer
1
Multiparameter Flow Cytometry Analysis
2
FCM and ELISA Analysis of CD4+ T Cells
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For FCM, CD4+T cells (1 × 105/500 µL) were stimulated for 6 hours at 37°C with 1 µL of a cell stimulation cocktail containing brefeldin A (BioLegend) and then cultured with surface antigen antibodies for 30 minutes at 4°C in the dark. Following washes with PBS, cells were fixed and permeabilized for 60 minutes with fixation/permeabilization diluent and concentrate (BioLegend) or intracellular fixation buffer (BioLegend). At 4°C in the dark, intracellular marker staining was performed for 30 minutes. FCM was conducted with the following antibodies from BioLegend: anti-human Foxp3-PE (clone 259D) and IL-17-PE (clone BL168). CytExpert software was used to analyze the results. For ELISA, CD4+T cells were seeded in 24-well plates (1 × 105 cells/well), and their supernatants were collected after 72 hours. IL-10 and IL-17 protein expression levels were measured using a DuoSet ELISA Development Kit (R&D Systems, Minneapolis, MN, USA).
3
Flow Cytometric Analysis of Cell Degranulation
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For the detection of degranulation, cells were stimulated for 4 h with PBDu and ionomycin in the presence of an anti-CD107a-BV421 (Biolegend, clone 1D4B, 121617) antibody. Next, cells were stained with anti-CD107a-PE-Cy7 (Biolegend, clone 1D4B, 121619) and other surface antibodies, followed by intracellular granzyme B and perforin staining, by using the intracellular fixation buffer (Biolegend, 420801) and permeabilization buffer (Biolegend, 421002) as per manufacturer instructions.
4
Cytokine Production in Activated Leukocytes
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Whole blood (150 μL) was lysed with ACK buffer. One million cells were seeded in a 24-well plate and cultured with Brefeldin A solution for 20 minutes at 37°C. Cells were then stimulated with 250 ng/mL of LPS for 10 hours at 37°C. After stimulation, cells were collected and washed with PBS prior to cell surface staining with viability dye–APC-Cy7, CD45-PE-Cy7, CD66b-PE, and CD16-APC for 30 minutes at 4°C. Cells were washed again with PBS before fixation (BioLegend intracellular fixation buffer) for 30 minutes at RT. Cells were washed twice with permeabilization buffer (BioLegend perm wash buffer). Cells were incubated with TNF-α–PerCP-Cy5.5 and IL-6–FITC overnight prior to washing and analysis on BD FACSCanto.
5
Cytokine Profiling of Immune Cells
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The lymph node and spleen derived single cell suspensions were stimulated with 50 ng mL -1 PMA, 500 ng mL -1 ionomycin in the presence of 0.5 μL brefeldin A (to prevent the cells from secreting cytokines) (Biolegend, Switzerland) for 4 h at 37 °C, 5% CO 2 . The cell suspensions were washed with 0.5 mL of 1% fetal calf serum and centrifuged at 300 g for 5 min. Cells were stained according to manufacturer's instructions with antibodies for CD3 (PeCy7 anti-mouse CD3ε, Biolegend, CH) and CD4 (Pacific blue anti-mouse CD4, Biolegend, Switzerland). Following fixation with intracellular fixation buffer (Biolegend, CH) and permeabilization of the cells with Permeabilization buffer (Biolegend, CH), the lymph node cells were stained for IL-4 (anti-mouse IL-4 APC, eBiosciences, Switzerland), IL-10 (anti-mouse IL-10 PE, eBiosciences, CH), IL-17A (anti-mouse IL-17A Alexa Fluor 488, eBiosciences, Switzerland) and IFN-γ (anti-mouse IFN-γ PerCP-Cy5.5, eBiosciences, CH), according to manufacturer protocols. The cells were washed and re-suspended in MACS buffer (Mitenyi Coulter, Switzerland) and a Gallios flow cytometer (Beckman Coulter, Switzerland) was used to measure cellular fluorescence with data processing by Kaluza software (Beckman Coulter, Switzerland). The results were expressed as percentage of the total cell number.
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