For surface staining alone, splenocytes were resuspended at a concentration of 1 × 106 cells/mL in staining buffer (PBS 1x and 1% FBS). Fc receptors were blocked by the addition of unlabeled anti-CD16/32 (Fc block; BD Pharmingen, San Diego, CA, USA). The leukocytes were then stained for 20 min at 4 °C with the optimal dilution of FITC anti-mouse CD3 and APC anti-mouse CD4 antibody (BD, Pharmingen). Cells were washed twice with staining buffer, resuspended in 100 µL, and an equal volume of 2% formalin was added to fix the cells. After that, cells were treated with permeabilization buffer and intracellular IFNγ and IL-7A cytokines were identified with PE anti-mouse IL-17A and PE-Cy7- anti-mouse IFNγ respectively (BD, Pharmingen). The stained cells were analysed with a BDAccuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA).
Pe anti mouse il 17a
Manufactured by BD
The PE anti-mouse IL-17A is a laboratory reagent used to detect and quantify the presence of interleukin-17A (IL-17A) in mouse biological samples. It is a fluorescently-labeled antibody that binds specifically to the IL-17A protein, allowing for its identification and measurement.
Automatically generated - may contain errors
Lab products found in correlation
Fitc anti mouse cd4, by BD (2 mentions)
Fc block, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Anti mouse cd3 fitc, by BD (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Fitc anti mouse ifn γ, by BD (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Percp cy5.5 anti mouse cd4, by BD (1 mentions)
Apc anti mouse il 4, by BD (1 mentions)
Alexa fluor 647 anti mouse foxp3, by BD (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Rnasin ribonuclease inhibitor, by Promega (1 mentions)
Sodium fluorescein, by Merck Group (1 mentions)
Pe anti mouse foxp3, by BD (1 mentions)
Anti mouse ifn γ apc, by BD (1 mentions)
Anti mouse rat foxp3 staining set apc, by Thermo Fisher Scientific (1 mentions)
Montelukast, by Merck Group (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Milli q water, by Merck Group (1 mentions)
9 protocols using pe anti mouse il 17a
1
Cytokine Profiling of Mouse Splenocytes
2
Th Cell Phenotyping in Murine Spleen
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenic CD4+ cells from approximately 28-day-old mice were obtained using the CD4+ T cell isolation kit (Miltenyi Biotec, 130-104-454) or by sorting with the BD FACSAria II cell sorter. CD4+ cells were processed using the Mouse Th1/Th2/Th17 (BD Biosciences, 560758) and Th17/Treg (BD Biosciences, 560767) phenotyping kits. Cells were cultured for 3–4 hours with or without 50 ng/mL PMA and 1 μg/mL ionomycin and then harvested for RNA-Seq or flow analysis. The following antibodies were used: PerCP-Cy5.5 anti–mouse CD4 (BD Biosciences, 550954), PE anti–mouse IL-17a (BD Biosciences, 559502), FITC anti–mouse IFN-γ (BD Biosciences, 554411), APC anti–mouse IL-4 (BD Biosciences, 554436), and Alexa Fluor 647 anti–mouse FOXP3 (BD Biosciences, 560402).
3
Murine T Cell Differentiation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Montelukast were from Merck. M-MLV Reverse Transcriptase and RNasin Ribonuclease inhibitor were from Promega (Fitchburg, WI). SYBR Green JumpStart™ Taq ReadyMixTM kit and sodium fluorescein were from Sigma (St.Louis, MO). Fluorescein isothiocyanate (FITC) anti-mouse CD45, FITC anti-mouse CD8a, phycoerythrin (PE) anti-mouse CD45R (B220) and APC anti-mouse/rat Foxp3 staining set were purchased from eBioscience (San Diego, CA). PE-Cy7 anti-mouse CD4, PE anti-mouse IL17A, APC anti-mouse IFN-γ, PE anti-mouse Foxp3 and BD Cytofix/Cytoperm kit were purchased from BD bioscience (Franklin Lakes,NJ). IL-17A, IFN-γ, TGF-β, TNF-α, IL-1β ELISA kit were from Dakewe (Shenzhen, China).
4
Investigating Immunomodulation in Murine Models
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Female 8-12 weeks old wild-type (WT) C57BL/6 mice, TNFR1−/− or TNFR2−/− mice with C57BL/6 background were used in this study. All mice were housed and treated with the approval of the Animal Research Ethics Committee of the University of Macau. PerCP-Cy5.5 anti-mouse TCRβ (clone: H57-597), PE anti-mouse IL-17A (clone: TC11-18H10), and PE anti-mouse CD120b/TNFR2 (clone: TR75-89) were purchased from BD Pharmingen (San Diego, CA). PE-Cy7 anti-mouse CD4 (clone: GK1.5), APC anti-mouse/rat Foxp3 (clone: FJK-16s), and FITC anti-human Ki-67 Monoclonal Antibody (clone: B56) were purchased from eBioscience. Hoechst 33342 was purchased from Invitrogen (Carlsbad, CA, USA). Anti-17 antibody (ab79056) and goat antirabbit IgG H&L (Alexa Fluor 488; ab150077) were purchased from Abcam, UK. TET (Cat: T2695) was purchased from Sigma-Aldrich. IMQ cream (5% w/w) was purchased from Health Care Limited (Loughborough, UK). Tacrolimus ointment (0.1% tacrolimus, Japan) was purchased from Astellas Pharma Tech Co., Ltd. Kolliphor® HS 15 and Kollisolv® PEG 400 were gift samples kindly offered by BASF Advanced Chemicals Co., Ltd, China. Labrafil M 1994 CS was a free sample from Gattefossé Co., Cedex, France. Milli-Q water was from a Millipore Direct-Q ultra-pure water system (Millipore, Bedford, USA). Carbopol 974N was purchased from Shanghai Chineway Pharmaceutical Technology Co., Ltd.
5
Th17 Cell Phenotypic Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, the cells were fixed and permeabilized for 30 min at 4°C using the CytoFix/CytoPerm kit (cat. no. 554714; BD Biosciences), washed with PBS and stained with PE anti-mouse IL-17A (cat. no. 559502; clone, TC11-18H1; BD Biosciences), BD Transduction Laboratories™ FITC mouse anti-Ki-67 (cat. no. 612472; clone, 35/Ki-67; BD Biosciences), BD Pharmingen™ FITC rabbit anti-active caspase-3 (cat. no. 560901; clone, C92-605; BD Biosciences), BD Pharmingen™ FITC hamster anti-mouse Bcl-2 (cat. no. 556357; BD Biosciences) or with its isotypic control antibody for 50 min at 4°C. Live lymphocytes were gated according to forward and side scatter, then by CD4 expression. BD Pharmingen™ PE-Cy™5 mouse IgG1 isotype control (cat. no. 550618; clone, MOPC-31C; BD Biosciences) was used as the isotype control. According to this gating strategy, CD4+ cells were considered helper T (Th) cells; CD4+IL-17+ cells were defined as Th17 cells. Ki-67, caspase-3 and Bcl-2 expression in Th17 cells were measured as described. Cells were detected by a flow cytometer (FACS Calibur; BD Biosciences) and analyzed using FlowJo 7.6.5 software (FlowJo LLC).
6
Flow Cytometric Analysis of Immune Cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We labeled cells with antibodies against markers: anti‐mouse CD11b‐PE (clone: M1/70; BioLegend), anti‐mouse Gr‐1‐FITC (clone: RB6‐8C5; BioLegend), anti‐mouse Ly6G‐FITC (clone: 1A8‐Ly6g; eBioscience), anti‐mouse Ly6C‐APC (clone: HK1.4; eBioscience), anti‐mouse CD4‐FITC (clone: GK1.5; BD Biosciences), anti‐mouse CD4‐APC (clone: GK1.5; eBioscience), anti‐mouse IL‐17A‐PE (clone: TC11‐18H10; BD Biosciences), anti‐mouse Foxp3‐PE (clone: FJK‐16s; eBioscience), anti‐mouse CD124‐PE/Cy7 (IL‐4Rα, clone: I015F8; BioLegend), anti‐mouse CD119‐PE (IFN‐γR1, clone: 2E2; eBioscience), anti‐mouse iNOS‐APC (clone: CXNFT; eBioscience), anti‐mouse CCR2‐APC (clone: 475301; R&D Systems), and anti‐mouse CD197‐PE/Cy7 (CCR7, clone: 4B12; eBioscience). MDSCs and T lymphocytes were surface stained after 30 min at 4°C incubation with relevant antibodies shielded from light. CD4+ T cells were stained with anti‐IL‐17A after being stimulated with the Cell Stimulation Cocktail (eBioscience) for 4–6 h to measure intracellular cytokine production. Following incubation in 1× Fixation and Permeabilization Solution, CD4+ T was stained with Foxp3 antibody for Treg detection. Data were acquired on a BD FACSCalibur™ flow cytometer (BD Biosciences) and analyzed with FlowJo version 7.6.1 (Treestar).
7
Intracellular Cytokine Staining for IL-17A
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To dye IL-17A, an intracellular CK, mouse peripheral blood was immersed in a 1x cell-stimulating cocktail comprising protein transport inhibitors(500xX) for 6 h. After harvesting the cultures, they were subjected to immobilization and permeabilization using IC fixation buffer (cat. no. 00-822) and permeabilization buffer (cat. no. 00-8333, Thermo Fisher Scientific, Shanghai, China), respectively. This was followed by cell staining with Anti-Mouse CD4 PE-CY-5 (cat. no. 553050, BD, New Jersey, USA) and Anti-Mouse CD25 BB515 (BD, cat. no. 564424) (30 min, 2-8°C). The cells were centrifuged (350 × g, 5 min) after one rinsing with a staining buffer (2 ml). Then, intracellular staining for Foxp3 was carried out. After completing cell surface staining, the residual stain buffer was removed and the cell mass was briefly loosened by vortexing. Then, came the addition of Fix/Perm Buffer working solution to each tube for cell mass resuspension by vortexing for 3 secs. or so. Thereafter, the specimens were treated with light-tight cultivation (2-8°C, 40-50 min). Finally, stained cells with Anti-Mouse IL-17A PE (BD, cat. no. 559502) and ANTI-M/R Fox3 PE-Cy-7(BD, cat. no. 25-5773-82).
8
Comprehensive Murine Immune Cell Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell suspensions from the spleens of mice were isolated with 40 μm cell strainers. Cells were incubated with antibody cocktails containing anti–mouse CD16/CD32 Fc block (catalog 553142, BD Biosciences) at 4°C for 30 minutes. The antibodies were anti–mouse CD4–FITC (catalog 553729), anti–mouse CXCR5–PE-Cy7 (catalog 560671), anti–mouse PD-1–PE (catalog 551892), anti–mouse IFN-γ–PE (catalog 562020), anti–mouse IL-17A–PE (catalog 561020), anti–mouse CD19–V450 (catalog 560375), anti–mouse CD80–APC (catalog 560016), anti–mouse PD-L2–PE (catalog 557796), anti–mouse CD138–PE (catalog 561070), and anti–mouse TACI–Alexa Fluor 647 (catalog 558453) (all from BD Biosciences). Data were acquired using a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using FlowJo software (version 10.8, FlowJo).
9
Cytokine Expression in Stimulated Lung Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated lung cells were stimulated for 4-5 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Sigma-Aldrich), followed by monensin (BD Biosciences) treatment for the last 2-3h of cultivation at 37°C and 5% CO 2 . Stimulated lung cells were incubated with FITC-conjugated antibodies for CD4 (clone GK1.5), fixed and permeabilized with BD GolgiPlug (BD Biosciences), and incubated with anti-mouse IL-17-A-PE, IL-4-PE, or IFN-g-PE (BD Biosciences) for 30 min at 4°C. Finally, cells were washed, fixed with 1% PFA, and analyzed using an Accuri C6 flow cytometer. Results were expressed as percentages of positive cells.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!