For identifying MDSCs, CD45+ cells magnetically sorted from the co-culture of PBMCs with uninfected/infected Huh7.5.1 cells were blocked with FcR blocking reagent (Miltenyi) and stained with the live/dead marker DAPI (Life Technologies), anti-CD33, -CD11b, and -HLA-DR (all from BD Pharmigen). For detecting intracellular arginase-1 production, CD33+ cells were magnetically sorted from co-cultures with NK cells and stained for MDSC surface markers. The cells were then fixed and permeabilized by Cytofix/Cytoperm (BD biosciences) and stained with the MDSC markers described above and anti-Arginase-1 (R&D Systems). Aqua live/dead stain (Life Technologies) was included to analyze cell viability. All stained cells were run on BD FACSCantoII (BD Biosciences) and analyzed using FlowJo software.
Anti arginase 1
Manufactured by R&D Systems
Anti-arginase-1 is a laboratory product that functions as an antibody against the arginase-1 protein. Arginase-1 is an enzyme involved in the urea cycle. This antibody can be used to detect and study the expression of arginase-1 in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Aqua live dead stain, by Thermo Fisher Scientific (1 mentions)
Anti inos, by R&D Systems (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Fixation and permeabilization solution kit, by BD (1 mentions)
Anti cd127 a019d5, by BioLegend (1 mentions)
Human fcr binding inhibitor, by Thermo Fisher Scientific (1 mentions)
Anti cd40 5c3, by BioLegend (1 mentions)
Anti mmp 9, by R&D Systems (1 mentions)
Anti cd45 hi30, by Thermo Fisher Scientific (1 mentions)
3 protocols using anti arginase 1
1
Identifying Myeloid-Derived Suppressor Cells
2
Western Blot Analysis of Immunomodulatory Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly isolated spDC, TIDC, or murine normal hepatocytes were lysed at 4°C for 20 min in a RIPA buffer containing protease inhibitors (2.5 μg/mL pepstatin, 10 μg/mL aprotinin, 5 μg/mL leupeptin, and 0.1 mM PMSF). After centrifugation, protein concentration in the supernatant was determined using a Bio-Rad protein assay (Hercules). Thirty micrograms of proteins was separated by SDS-PAGE (12% polyacrylamide gel for IDO and arginase-1 detection and 7% for inducible NOS (iNOS) detection). Proteins were electrotransferred onto a nitrocellulose membrane. The membrane was blocked in Tris Buffered Saline (TBS) with 1% Tween-20 and 5% skim milk and incubated overnight (4°C) with anti-mouse-IDO monoclonal antibody (1 : 5000, Enzo Life Sciences), anti-arginase-1 (1 : 2000, R&D Systems), and anti-iNOS (1 : 500, R&D Systems). The membrane was washed and incubated (room temperature, 2 h) with HRP-conjugated secondary antibody. Immunoblots were then developed using an enhanced chemiluminescence (ECL) reagent kit from Santa Cruz Biotechnology, according to the manufacturer's protocol.
3
Characterizing Immune Cell Phenotypes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human FcR Binding Inhibitor (eBioscience) was added to the samples (1:200) and incubated for 20 min on ice to block non-specific Fc-receptor binding. After, cells were washed with FACS buffer and stained with surface antibodies, which included anti-CD45 (HI30, eBioscience); anti-CD40 (5C3, BioLegend); and anti-CD127 (A019D5, BioLegend), at room temperature for 15 min in the dark. Samples were washed twice with FACS buffer. Intracellular staining with anti-CD68 (eBioY1/82A, eBioscience), anti-arginase-1 (R&D systems), and anti-MMP9 (R&D systems) was performed using the Fixation and Permeabilization Solution kit (BD Biosciences). Dead cells were excluded using 7-AAD staining. Samples were run on BD LSRII flow cytometer and data were analyzed using FACSDiva software and FlowJo.
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!