Dendritic Cell Protocols by Elodie Segura, Nobuyuki Onai

By Elodie Segura, Nobuyuki Onai

The 3rd variation of this quantity is geared toward offering either newbies and more matured researchers a call of ways to isolate and study dendritic cells(DC). An introductory evaluate presents an outline of modern advances within the characterization of DC subsets in mouse and human. whereas extra chapters offer the way to tradition human and mouse dendritic cells, protocols for the isolation of dendritic cells, the isolation of dendritic telephone progenitors from mouse, and the purification of dendritic cells from human blood. Written within the hugely winning Methods in Molecular Biology series structure, chapters comprise introductions to their respective issues, lists of the required fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and tips about troubleshooting and warding off recognized pitfalls.

 

Authoritative and cutting-edge,  Dendritic phone Protocols, 3rd version aims to make sure winning ends up in the extra research of this very important field.

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2 Freezing MuTu DC Lines 1. Place the freezing vials at 4 °C 1 h before use. 2. Dissociate the cells by incubating them for 10 min in cold cell dissociation buffer. 3. Centrifuge the cells at 360 × g for 5 min. 4. Label the precooled freezing vials (see Note 16). 5. Gently dissociate the dry pellet. 6. Resuspend the cells at 3 × 106/mL in ice-cold freezing medium. 7. Transfer the cells to the vials (1 mL per vial). Close the tubes well. 8. Place the tubes quickly in the freezing container and place the container at −70 °C (see Note 17).

Ahrens S, Zelenay S, Sancho D, Hanc P, Kjaer S, Feest C, Fletcher G, Durkin C, Postigo A, Skehel M, Batista F, Thompson B, Way M, Reis e Sousa C, Schulz O (2012) F-actin is an evolutionarily conserved damage-associated molecular pattern recognized by DNGR-1, a receptor for dead cells. Immunity 36(4): 635–645 11. Sancho D, Joffre OP, Keller AM, Rogers NC, Martinez D, Hernanz-Falcon P, Rosewell I, Reis e Sousa C (2009) Identification of a dendritic cell receptor that couples sensing of necrosis to immunity.

3. FACS buffer: PBS, 1 mM EDTA, 10 mM HEPES. 4. Staining buffer (SB): FACS buffer, 2 % FCS. 5. Human TruStain FcX™ (Fc Receptor Blocking Solution, BioLegend). 6. , 50 μL TruStain FcX™ for 1 mL SB). 7. LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Invitrogen). 8. 5 % paraformaldehyde working solution: prepare 4 % (w/v) stock solution in PBS, adjusted to pH 7, according to manufacturer’s instructions. Stock solution should be aliquoted in 10 mL volumes in 15 mL polypropylene tubes and frozen at −20 °C.

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