After cell viability staining with ZombieDye NIR (Biolegend, CA, USA), cell suspensions were stained in PBS 1 × 5% heat‐inactivated FBS (Gibco, USA) with the following antibodies: CD3e (clone 145‐2c11), CD4 (clone RM4‐5), CD11b (clone M1/70), CD11c (clone N418), CD25 (clone PC61), CD45.1 (clone A20), CD45.2 (clone 104), CD206 (clone C068C2), I‐A/I‐E (clone M5/114.15.2), Ly‐6C (clone HK1.4), Ly‐6G (clone 1A8), NK1.1 (clone PK136), Nrp1 (clone 3E12), all from Biolegend, CA, USA. Surface staining was performed for 30 min at 4°C. Intracellular staining was performed with anti‐Foxp3 (clone FJK‐16s) and the Fixation/Permeabilization Staining Kit (all from eBioscience, CA, USA), following manufacturer's instruction. For cytokine expression analysis, cells were activated with 50 ng/ml PMA (Sigma) and 1 μg/ml Ionomycin (Sigma) in RPMI containing 10% FBS and Brefeldin‐A (eBioscience, CA) for 4 h. Cells were washed, stained for surface markers, and treated with the Fixation/Permeabilization Staining Kit (all from eBioscience, CA, USA), following manufacturer's instructions. Intracellular cytokine staining was performed with the following antibodies: IFN‐γ (clone XMG1.2), IL‐17A (clone TC11‐18H10.1, both from Biolegend). Cells were analysed on FACSCanto II (BD Biosciences, USA) or FACS‐sorted in a FACSAria II (BD Biosciences, USA). Data analysis was performed on FlowJo software (CA, USA).
Fixation permeabilization staining kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Fixation/Permeabilization Staining Kit is a laboratory product designed for the preparation of samples for flow cytometry analysis. The kit provides the necessary reagents to fix and permeabilize cells, allowing for the intracellular staining of target proteins or molecules.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by Thermo Fisher Scientific (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Anti foxp3 clone fjk 16s, by Thermo Fisher Scientific (1 mentions)
Zombiedye nir, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
70 μm cell strainer, by Merck Group (1 mentions)
Facscanto cytometer, by BD (1 mentions)
Anti cd4 gk1, by BioLegend (1 mentions)
Anti foxp3 mf 14, by BioLegend (1 mentions)
Cd25 pc61, by BioLegend (1 mentions)
Cd4 cd25, by BD (1 mentions)
Zombie uv fixable viability kit, by BioLegend (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Ifn γ pe, by BD (1 mentions)
Il 4 pe cy7, by BD (1 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions)
4 protocols using fixation permeabilization staining kit
1
Multiparametric Flow Cytometry Analysis
2
Flow Cytometric Analysis of Lymphocytes
3
Profiling Immune Cells in Periodontal Tissues
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Single-cell suspensions were obtained from cervical lymph nodes that drain periodontal tissues, and spleens using 70 μm cell strainers (Sigma-Aldrich) rinsed-out with phosphate-buffered saline (PBS) containing 5% fetal bovine serum (FBS). For cytokine detection, 2–4 × 106 cells were incubated for 4 h in RPMI -1640 (Gibco) supplemented with 10% FBS, 1% penicillin–streptomycin (Sigma), Brefeldin A (eBioscience), 50 ng/ml PMA (Sigma), and 1 μg/ml Ionomycin (Sigma). Cells were then washed with PBS and stained with the zombie UV™ Fixable Viability Kit (Biolegend) for 30 min in the dark. The extracellular staining was performed in PBS containing 5% FBS, using the following antibodies: anti-CD4 (GK1.5, Biolegend) and CD25 (PC61, Biolegend), for 30 min at 4 °C in the dark. The intracellular staining was done using a Fixation/Permeabilization staining kit following the manufacturer instructions (eBioscience) and using the following antibodies: anti-Foxp3 (MF-14, Biolegend), Rorγt (Q21-559, eBiosciences), and IL-17A (9B10, Biolegend). Cells were analyzed on a BD FACSCanto cytometer (BD Biosciences) using a sequential gating strategy according to the FSC/SSC and SSC/SSC parameters, live/dead staining, and CD4/CD25 markers. Data analysis was completed using the FlowJo software (version 10.6.2). For each animal, the experiments were performed separately.
4
Multiparametric Flow Cytometry Analysis of Murine Splenocytes
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The method of mouse euthanasia was described previously. Single cells from the spleen were suspended in RPMI 1640 culture medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA) and Cell Stimulation Cocktail (eBioscience, San Diego, CA, USA) and then cultured in 6-well plates for 5 hours in a 37°C/5% CO2 incubator.
Lymphocytes were separated from the spleen by passing through a 70 μm nylon mesh. Erythrocytes were lysed with ammonium chloride buffer, after which the remaining cells were washed and counted using a hemocytometer. One million cells were suspended in staining buffer with the following fluorochrome-conjugated antibodies: CD4-FITC (mAb; BD, Franklin Lakes, NJ, USA), CD25-APC (mAb; BD, Franklin Lakes, NJ, USA), IL-4-PECy7 (mAb; BD, Franklin Lakes, NJ, USA), IFN-γ-PE (mAb; BD, Franklin Lakes, NJ, USA), and Foxp3-PE (mAb; BD, Franklin Lakes, NJ, USA). Intracellular Foxp3, IL-4, and IFN-γ within lymphocytes were stained using a fixation/permeabilization staining kit (eBioscience, San Diego, CA, USA). All samples were analyzed using a FACSCanto II flow cytometer (BD, Franklin Lakes, NJ, USA) and analyzed using Flow Jo analysis software (Tree Star Inc.).
Lymphocytes were separated from the spleen by passing through a 70 μm nylon mesh. Erythrocytes were lysed with ammonium chloride buffer, after which the remaining cells were washed and counted using a hemocytometer. One million cells were suspended in staining buffer with the following fluorochrome-conjugated antibodies: CD4-FITC (mAb; BD, Franklin Lakes, NJ, USA), CD25-APC (mAb; BD, Franklin Lakes, NJ, USA), IL-4-PECy7 (mAb; BD, Franklin Lakes, NJ, USA), IFN-γ-PE (mAb; BD, Franklin Lakes, NJ, USA), and Foxp3-PE (mAb; BD, Franklin Lakes, NJ, USA). Intracellular Foxp3, IL-4, and IFN-γ within lymphocytes were stained using a fixation/permeabilization staining kit (eBioscience, San Diego, CA, USA). All samples were analyzed using a FACSCanto II flow cytometer (BD, Franklin Lakes, NJ, USA) and analyzed using Flow Jo analysis software (Tree Star Inc.).
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