Recombinant proteins of mouse sortilin and cytokines except for mouse IFN-α were purchased from R&D. GST-fused mouse IFN-α was purified as follows: the plasmid pGEX-IFNA2 was introduced into Escherichia coli strain Rosetta 2(DE3) (Novagen, Madison, WI). Plasmid-bearing cells were initially grown aerobically at 37 °C on L medium46 containing 100 μg/ml ampicillin. Expression of recombinant proteins was then induced by the addition of 0.2 mM IPTG at 20 °C for 18 h. Cell lysates were prepared with FastBreak Cell Lysis Reagent (Promega, Madison, WI). Isolated supernatants were purified with a GSTrap 4B column (GE Lifesciences). Eluted protein fractions were then desalted through a HiTrap Desalting column (GE Lifesciences) and fractions containing purified proteins were concentrated with Vivaspin 20 spin columns (GE Lifesciences). Concentrated proteins were stored at −80 °C until use. Surface plasmon resonance analysis was performed as described47 (link) on a Biacore X instrument (GE Lifesciences). Kinetic parameters were determined using BIAevaluation software.
Mouse ifn α
Manufactured by R&D Systems
Sourced in United States
Mouse IFN-α is a recombinant protein that represents the interferon-alpha subtype found in mice. Interferons are a class of signaling proteins involved in the immune response against viral infections and other biological activities.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap desalting column, by GE Healthcare (1 mentions)
Gstrap 4b column, by GE Healthcare (1 mentions)
Fastbreak cell lysis reagent, by Promega (1 mentions)
Biacore x instrument, by GE Healthcare (1 mentions)
Hitrap, by Cytiva (1 mentions)
Mouse il 2, by Thermo Fisher Scientific (1 mentions)
Mouse il 12, by R&D Systems (1 mentions)
Facsarray, by BD (1 mentions)
Fcap array software, by BD (1 mentions)
Ficoll, by Merck Group (1 mentions)
Macs separation, by Miltenyi Biotec (1 mentions)
Ammonium chloride potassium lysis buffer, by Avantor (1 mentions)
4 protocols using mouse ifn α
1
Purification and Analysis of Mouse IFN-α
2
Murine NK Cell Activation Assay
3
Quantifying pDC Cytokine and Chemokine Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
pDCs were cultured at 2.5×106 cells/mL with IMQ and CpG in RPMI medium supplemented with 10% FBS, 1% Pen/Strep, 1% nonessential amino acids, 1% sodium pyruvate, and 0.1% β-mercaptoethanol. Supernatant were collected after 36 hours, and analyzed with the following ELISA kits: mouse IFN-α (R&D Systems, USA), and mouse Granzyme B (Elabscience, USA). The levels of cytokine IL-12p70, TNF-α, and IL-6, as well as the levels of chemokine CCL3 and CCL5, in supernatant from resting or activated pDCs were analyzed using a cytometric bead array (CBA; BD, USA). The levels of chemokine CCL3 and CCL5 in serum from mice treated with resting or activated pDCs were analyzed using CBA. Briefly, 50 μl of samples or recombinant standards were added to 50 μl of mixed capture beads, and incubated for 3 hours with 50 μl of phycoerythrin-conjugated detection antibodies (Ab-PE) to form sandwich complexes. After washing to remove unbound Ab-PE detection reagent and performing FACS analysis using FACSArray (BD, USA), results were generated using FCAP Array software (BD, USA).
4
Generating Stable Cell Lines Expressing Sialidases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CHO-K1 cells were kindly provided by Dr. J. D. Esko (University of California, San Diego, CA, USA) and were transfected or transduced to obtain stable cell lines expressing hSn (hSn + -CHO) and mSn (mSn + -CHO) [37] . These cells were subcultured in RPMI with 10% heat inactivated fetal bovine serum (iFBS). Primary mouse bone marrow macrophages were collected in RPMI medium. After removing red blood cells with an ammonium-chloridepotassium lysis buffer (VWR and Janssen Chimica), macrophages were seeded in RPMI with 10% iFBS, 1% non-essential amino acids, 1% sodium pyruvate, 1% glutamine and with addition of L929 supernatant and 500 U/ml mouse IFN-α (R&D) in the medium. L929 supernatant was collected from cells kindly provided by Dr. C. Uyttenhove (Ludwig Institute for Cancer Research, Brussels, Belgium). Human monocytes were obtained from blood of healthy volunteers from the Belgian Red Cross-Flanders. The monocellular cells were isolated from the blood using Ficoll (Sigma-Aldrich). CD14 + monocytes were isolated using MACS separation following manufacturer's instructions (Miltenyi Biotec). The cells were seeded in RPMI with 10% heat inactivated human serum, 1% non-essential amino acids, 1% sodium pyruvate and 1% glutamine. After two days in culture, 50 ng/ml human IFN-α was added for three days to obtain hSn-positive macrophages.
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!