For the analysis of Tregs and MSCs in lung lymph nodes, the pulmonary hilar lymph nodes were collected and teased apart into a single cell suspension by pressing with the plunger of a 3 ml syringe. Harvested lung tissues were minced and incubated for 45 min at 37°C in DMEM supplemented with 10ug/ml DNase I and 1 mg/ml collagenase D (Sigma Aldrich) in a shaking water bath. After that, the digested lung tissue was passed through a 70-um nylon strainer (BD Biosciences) to obtain single cell suspensions. Red blood cells were lysed by ACK lysis buffer. For Tregs, the cells were first stained with anti-CD4-FITC (RM4–5, eBioscience) and anti-CD25-PE antibodies (PC61.5, eBioscience), followed by intracellular staining with FoxP3-APC (FJK-16s, eBioscience) or APC-conjugated rat IgG2a isotype control (eBioscience). For MSCs, cells were stained with Sca-1, CD29, CD45, and CD11b fluorochrome conjugated antibodies (eBioscience). These samples were analyzed on a FACSCalibur flow cytometer (BD Biosystems).
Anti cd4 fitc rm4 5
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-CD4-FITC (RM4-5) is a fluorescently-labeled antibody that binds to the CD4 cell surface marker. It is designed for use in flow cytometry applications to identify and quantify CD4+ T cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd62l apc mel 14, by Thermo Fisher Scientific (3 mentions)
Anti cd44 pe im7, by BioLegend (3 mentions)
Foxp3 apc fjk 16s, by Thermo Fisher Scientific (2 mentions)
Anti cd25 pe antibodies pc61, by Thermo Fisher Scientific (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Anti cd25 pe cy5 pc61, by Thermo Fisher Scientific (2 mentions)
Anti cd28, by BD (2 mentions)
Pan t cell isolation kit 2, by Miltenyi Biotec (2 mentions)
Collagenase d, by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ apc xmg1.2, by Thermo Fisher Scientific (1 mentions)
Anti cd8 percp cy5 5 53 6, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
6 protocols using anti cd4 fitc rm4 5
1
Analysis of Tregs and MSCs in Lung Lymph Nodes
2
Tregs and TβRI/II Analysis in Lung and MSCs
3
Multiparameter Flow Cytometry for Mycobacterial Antigen-Specific T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Multiparameter flow cytometry was performed according to a standard protocol (36 (link), 37 (link)). For surface staining, single cell suspensions from spleen were prepared at the indicated time points. Freshly isolated lymphocytes were stimulated with ESAT6 (1.25 μg/ml), CFP10 (1.25 μg/ml), and HspX (2.5 μg/ml) for 4 h and subsequently incubated for 8 h with BD GolgiPlug (containing brefeldin A) at 37°C. Samples were stained with anti-CD4-FITC (RM4-5, eBioscience) and anti-CD8-PerCP-Cy5.5 (53–6.7, eBioscience). Then, cells were permeabilized using the BD Cytofix/Cytoperm kit according to the manufacturer's instructions and stained with anti-IFN-γ-APC (XMG1.2, eBioscience). The cell suspensions were analyzed by NovoCyte flow cytometer (ACEC Biosciences, Inc., Zhejiang, China).
4
Lipid Metabolism Regulates Regulatory T Cell Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleens from naïve C57Bl/6 mice were digested for 30 min in RPMI media containing DNaseI (10 mg/ml) and Liberase TL (1.67 Wünsch Units/ml). T cells were isolated by MACS using the Pan T cell isolation kit (Miltenyi). Cells were fluorescently stained with anti-CD4-FITC (RM4-5, eBioscience), anti-C25-PECy5 (PC61.5, eBioscience), anti-CD44-PE (IM7, BioLegend) and anti-CD62L-APC (MEL-14, eBioscience) to isolate naïve T cells with a cell sorter. Naïve T cells were cultured on 96 well plates together with plate-bound anti-CD3 (2 µg/ml, 145-2C11, BD Pharmingen) and anti-CD28 (2 µg/ml, 37.51, BD Pharmingen) and rhTGF-β1 (1 ng/ml) for Treg cell differentiation. The cells were additionally treated with solvent (PBS), LA (250µM) or LA+PA (PA 150µM) for 4 days and analyzed for the frequency of CD25+FoxP3+ cells in CD4+ viable lymphocytes with flow cytometry. In some experiments, cells were additionally treated with an IL-10 receptor blocker (1µg/ml, Biolegend).
5
Investigating TH17 Cell Differentiation Under ILA and Osmotic Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenic T cells were isolated by magnetic activated cell sorting using the “Pan T cell isolation kit II” (Miltenyi Biotec) according to the manufacturer’s instructions. Isolated T cells were collected and re-suspended in MACS buffer at 3·107 cells/ml. For APC free differentiation, cells were fluorescently stained for 30 min in an antibody cocktail containing anti-CD4-FITC (RM4-5, eBioscience), anti-CD44-PE (IM7, BioLegend), anti-CD62L-APC (MEL-14, eBioscience) and anti-CD25-PE-Cy5 (PC61.5, eBioscience) and subsequently purified by fluorescence activated cell sorting on MoFlo (Beckman-Coulter). Sorted naive T cells (CD4+CD62L+CD44lowCD25neg) were stimulated by plate-bound anti-CD3 (2 μg/ml, 145-2C11, BD Pharmingen) and anti-CD28 (2 μg/ml, 37.51, BD Pharmingen) in the presence of IL-6 (40 ng/ml) and rhTGF-β1 (2 ng/ml). To determine the influence of indole-3 lactic acid on TH17 cell differentiation, cells were cultured with vehicle (0.1% Ethanol) or 10-500 μM indole-3-lactic acid (ILA) for 96h under isotonic or hypertonic (+40 mM NaCl) conditions.
6
Investigating TH17 Cell Differentiation Under ILA and Osmotic Stress
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!