To study immune cell infiltrates in the postnatal heart, heart tissues were minced into small fragments and dissociated with 1:1 type II collagenase (1000 U/ml in PBS, Worthington) and dispase (11 U/ml in PBS, Gibco) at 37°C for 30 min. Enzymatic action was stopped by adding 10% FBS and the dissociated cells were washed twice with PBS. The dissociated single splenocytes or neonatal heart cells were removed from the contaminated erythrocytes by incubating with the red blood cell lysis buffer (eBiosciences) for 5 min; and were then blocked with 2% normal rabbit serum. Cells were subsequently stained with fluorochrome-conjugated antibodies against the following antigens: CD3, CD4, CD8, CD45, CD206, F4/80 or Ly6C (Biolegend or eBiosciences) at a dilution of 1:100, unless specified by the manufacturer, at 4°C for 30 min. Murine Treg of ICR mice were detected with the Treg staining kit according to manusfacturer's instructions (eBioscience). Cells were then washed three times with 2% FBS-containing PBS and analyzed on flow cytometer (BD FACSAriaTM Fusion). Propidium iodide (PI, BD) positive dead cells were excluded for live cell analysis/sorting; and FACS data were then analyzed with the FlowJo software (Tree star).
Treg staining kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Treg staining kit is a laboratory tool designed for the identification and analysis of regulatory T cells (Tregs). It provides the necessary reagents and protocols to stain and detect Tregs in various sample types using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Red blood cell lysis buffer, by Thermo Fisher Scientific (2 mentions)
Facsariatm fusion, by BD (2 mentions)
Dispase, by Thermo Fisher Scientific (2 mentions)
Collagenase type 2, by Worthington (2 mentions)
F4 80, by BioLegend (1 mentions)
Cd206, by BioLegend (1 mentions)
Facssuite, by BD (1 mentions)
Fcs express, by De Novo Software (1 mentions)
Anti cd16 32 2.4g2, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Mcd45, by BioLegend (1 mentions)
Fitc conjugated anti mouse cd3, by BD (1 mentions)
Apc conjugated anti mouse cd4, by BD (1 mentions)
Fcr block, by BD (1 mentions)
Pe conjugated anti mouse il 17a, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Cd8 bv510, by BioLegend (1 mentions)
Cell stimulation cocktail plus protein transport inhibitors, by BioLegend (1 mentions)
Intrasure kit, by BD (1 mentions)
12 protocols using treg staining kit
1
Isolation and Characterization of Cardiac Immune Cells
2
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were incubated with anti-CD16/32 (2.4G2) (BD Bioscience) to block FcγR II/III, and then stained with various conjugated antibodies as indicated. BD Cytofix/Cytoperm kit (BD Bioscience) was used for intracellular cytokine staining and Treg staining kit (e-Bioscience, San Diego, CA) was used for detection of Treg cells, following the manufacturer’s instructions Stained cells were analyzed by FACSVerse with FACS Suite (BD Bioscience) or FCS Express (De Novo Software, Los Angeles, CA) software.
3
Isolation and Characterization of Murine Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To collect splenocytes, the excised spleen was dissociated in PBS with a syringe plunger through a 40um cell strainer to obtain single cell suspension. To study immune cell infiltrates in the neonatal heart, heart tissues were minced into small fragments and dissociated with 1:1 type II collagenase (1000 U/ml in PBS, Worthington) and dispase (11 U/ml in PBS, Gibco) at 37°C for 30 minutes. Enzymatic action was stopped by adding 10% FBS and the dissociated cells were washed twice with PBS. The dissociated single splenocytes or neonatal heart cells were removed from the contaminated erythrocytes by incubating with the red blood cell lysis buffer (eBiosciences) for 5 minutes; and were then blocked with 2% normal rabbit serum. Cells were subsequently stained with fluorochrome-conjugated antibodies against the following antigens: mCD3, mCD4, mCD8, mCD31, mCD45, mF4/80, mLy6C or hCD2 (Biolegend or eBiosciences) at a dilution of 1:100, unless specified by the manufacturer, at 4°C for 30 minutes. Murine Treg were detected with the Treg staining kit according to manusfacturer's instructions (eBioscience). Cells were then washed three times with 2% FBS-containing PBS and analyzed on flow cytometer (BD FACSAriaTM Fusion). Propidium iodide (PI, BD) positive dead cells were excluded for live cell analysis/sorting; and FACS data were then analyzed with the FlowJo software (Tree star).
4
Intracellular Cytokine Analysis of T cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell suspensions were obtained from spleen of mice on indicated days postinfection. For intracellular cytokine staining, cells were stimulated for 5–6 h with PMA (50 ng/mL) and ionomycin (500 ng/mL) in the presence of brefeldin A (10 μg/ml). Cells were harvested, washed, and stained with surface molecule antibodies in the presence of FcR-Block (BD Bioscience). After the wash, cells were then fixed using CytoFix/CytoPerm buffer (BD Bioscience) and stained with antibodies against intracellular cytokines or isotype control on ice for 30 min. The following Abs were used for staining: FITC-conjugated anti-mouse CD3, PE-conjugated anti-mouse IL-17A, PE-Cy5.5-conjugated anti-mouse IFN-γ, and APC-conjugated anti-mouse CD4 (BD Bioscience). Treg cell staining was performed using a Treg staining kit according to the manufacturer’s instructions (eBioscience). Data were acquired on a FACS Calibur (BD Bioscience) and analyzed using Flowjo software (TreeStar).
5
Immune Cell Analysis in aGvHD Mouse Model
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
On day 8 after transplantation, the spleens of the aGvHD mouse model were crushed through 70-μm screens and the erythrocytes lysed. Single-cell suspensions were incubated in PBS containing the following fluorescently labeled antibodies: CD3 (PE), CD4 (APC/Cy7), and CD8 (BV510) (all from BioLegend, San Diego, CA, USA) at 4°C for 20 min. Apoptosis was assessed using PE Annexin V Apoptosis Detection Kit I (BD Biosciences, Franklin Lakes, NJ, USA). Chimerism was assessed by H2Kb (FITC; BioLegend) positive population. For intracellular cytokine staining, the cells were stimulated with cell stimulation cocktail plus protein transport inhibitors (BioLegend) for 4 h. Cells were then harvested and washed. After surface staining of CD3, CD4, and CD8, the cells were fixed and permeabilized using a fixation and permeabilization kit (BD IntraSure Kit) and stained for the intracellular cytokines IFNγ (PerCP) and IL4 (APC) (both from BioLegend). The CD4+CD25+Foxp3+ population assay was carried out using a mouse regulatory T cell (Treg) staining kit (eBioscience, San Diego, CA, USA). Data were acquired on a FACS Canto II (BD) system and were analyzed using FlowJo software.
6
Regulatory T Cell Depletion Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To establish Treg depletion, age-matched adult DEREG mice were injected i.p. with 1μg diptheria toxin (DT) or the inactive glu52 mutant as a control (Calbiochem) in 100μl of PBS for two successive days. The percent of FoxP3+CD4+ T cells in the spleen was assessed by flow cytometry using a Treg staining kit (eBiosciences) and determined to be 0.05% (not shown). This rebounded to 10% two days after the final DT injection. For WNV infection, littermate C57Bl/6 and DEREG adult and old mice were injected with 1μg DT in 100μl PBS on day -1 and day 0 of infection.
7
FoxP3 Treg Cell Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GolgiStop (BD PharMingen, San Diego, CA, USA), phorbol myristate acetate (PMA) and ionomycin were added during the last 4 h of each culture. A Treg staining kit (eBioscience) was used to stain FoxP3 in spleen and thymus cells according to the protocol provided by the manufacturer using anti-forkhead box protein 3 (FoxP3)-PE (eBioscience), anti-T-bet-PE (eBioscience) and anti-IFN-γ-APC (BD Pharmingen). Samples were analysed with fluorescence activated cell sorter (FACS)Calibur flow cytometer (Becton Dickinson, Mountain View, CA, USA), and data were analysed with FlowJo software (Tree Star, Ashland, OR, USA).
8
Thymocyte and Thymic Epithelial Cell Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For thymocyte staining, single-thymocyte suspensions were prepared from the thymi of mice using a 70-μm cell strainer. Samples were then stained with specific fluorochrome-conjugated antibodies of cell surface CD markers, indicated in each figure legend, and then fixed and permeabilized with fixation/permeabilization buffer (eBioscience Treg staining kit, #88-8115-40), per company’s instruction, followed by intracellular staining for PE-anti-FoxP3 (eBioscience Cat# 12-5773-82) or FITC-anti-FoxP3 (eBioscience Cat# 11-5773-82), and/or PE-anti-p-Zap70 (Cell Signaling, Cat# 14791, clone 65E4, recognizing phosphorylation at Tyr319/Syk [Tyr352]), PE-anti-Nur77 (eBioscience, Cat# 12-5965-82, clone 12.14), and AlexaFluor488-anti-GFP (BioLegend, Cat# FM264G). For TEC staining, the thymus was cut into pieces, then was digested with Collagenase-V/DNase-I, as per previously published methods [51 (link)], then stained with surface molecules: fluorochrome-conjugated anti-CD45 (clone 30-F11), -MHC-II (clone M5/114.15.2), -Ly51 (clone 6C3), and -EpCAM (clone G8.8), which were purchased from BioLegend, and FITC-anti-Ovalbumin from AbCam (Cat# ab85584). Flow cytometry was performed using an LSRII flow cytometer (BD Biosciences) with 6 colors, and data were analyzed using Flow-Jo software.
9
Multicolor Flow Cytometry Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All directly conjugated antibodies for flow cytometry were purchased from Biolegend. These include: B220, CD45, CD3, CD4, CD8, CD69, CD44, F4/80, Gr1, CD11b, CD11c, MHCII and CCR2. CD4+Foxp3+ regulatory T cells (Tregs) were identified using the commercially available Treg staining kit (eBioscience). To detect VISTA expression, 13F3 Ab from our laboratory was used.10 (link)
10
Comprehensive Immune Profiling of PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As described before [ 17 ], we isolated peripheral blood mononuclear cells (PBMC) by gradient centrifugation (Percoll) and stained PBMCs with a live/dead Fixable Viability Dye (Invitrogen, USA) and with antibodies (Biolegend, USA) against cell surface immune cell markers such as CD3, CD4, CD8, CD19, CD20, CD24, CD25, CD38, CD56, CD127, CD45RA and HLA-DR for 30 minutes at 4°C covered from light. Following cell surface staining, intracellular and or intranuclear immune markers CD152 (cytotoxic T-lymphocyte associated protein 4: CTLA-4) and FOXP3 antibody (eBioscience, USA) staining was performed after an incubation with FOXP3 fixation/permeabilization buffer and 1× permeabilization buffer (Treg staining kit, eBioscience, USA) as per supplier protocol. Stained cells were analysed by a 14-colour flow cytometry (Attune NxT). A minimum of 100 000 gated events were recorded. Written informed consent was obtained by parent/patient. Monitoring of lymphocyte subsets is as per standard (Koc University Ethics: 2019.255.IRB2.077). IL-6 and liver enzyme levels were collected from patient records.
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!