By Shimon Sakaguchi (auth.), George Kassiotis, Adrian Liston (eds.)
Driven via methodological good fortune in selecting trustworthy lineage markers, regulatory T cells have quick been famous because the such a lot various subset of immune regulators within the physique with serious capabilities in a wide range of immune responses. In Regulatory T Cells: tools and Protocols, specialists within the box to supply a suite of present recommendations to boost the research of regulatory T cells, together with using the IL-2 receptor alpha chain and different markers, in addition to the extra lately fascinating use of the transcription issue FoxP3. Divided into 3 sections, the ebook covers, in equivalent degree, in vitro, in vivo, and human reviews. Written within the hugely winning tools in Molecular Biology™ sequence structure, chapters comprise short introductions to their respective matters, lists of the mandatory fabrics and reagents, step by step, effectively reproducible laboratory protocols, and key tips about troubleshooting and keeping off identified methodological pitfalls. Authoritative and state-of-the-art, Regulatory T Cells: tools and Protocols distills the main important present innovations via numerous years of optimization and standardization so one can let trustworthy and reproducible use by means of either younger and skilled mobile and molecular immunologists.
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Additional resources for Regulatory T Cells: Methods and Protocols
As Tregs proliferate very poorly in vitro, they do not contribute significantly to cpm values. Control wells containing activated Tregs and no Tconv cells should have cpm values of less than 1,000, similar to that seen in wells containing unstimulated Tconv. Due to day to day or sample to sample variability, experimental replicates will often not result in identical cpm values. For this reason, a percent suppression (% supp) calculation assay can be graphed in order to depict many experiments with slightly (or significantly) different cpm values.
The lower part of the tube containing cells should remain immersed in water. Start to dilute out the PEG by adding the 10 ml of prewarmed DMEM, dropwise and gently swirling to mix the PEG with the DMEM. First, add 1 ml MEM over the course of 30 s, then add 2 ml more of MEM over the course of 30 s. Continue by adding slowly 3 ml MEM over the course of 30 s and finally add the remaining 4 ml MEM over the next 30 s. As medium is added to dilute PEG, try to minimize the shear forces due the fragile nature of the hybridomas at this time.
Slowly push 10 ml of perfusion solution through the heart. To further displace blood from the liver, push an additional 5 ml of perfusion solution through the liver via the portal vein at the base of the liver. After removing the gall bladder from the liver, crush the liver through a nylon 100 mm cell strainer into a 50-ml conical tube using 30 ml of balanced salts solution. Centrifuge for 10 min at 850 × g with no brake. Aspirate the supernatant and resuspend the pellet in 15 ml of 35% Percoll by vortexing.
Regulatory T Cells: Methods and Protocols by Shimon Sakaguchi (auth.), George Kassiotis, Adrian Liston (eds.)