Macrophages and Dendritic Cells: Methods and Protocols - download pdf or read online
By Georgia Schäfer, Gordon D. Brown* (auth.), Neil E. Reiner (eds.)
In gentle of the serious contributions of macrophages and dendritic cells to different inflammatory illnesses and to immunity and host safety, cutting-edge techniques to the research in their habit are crucial. In Macrophages and Dendritic Cells: equipment and Protocols, specialist researchers give a contribution laboratory protocols concerning those important mobile varieties performing at the junction of the innate and bought immune structures. the amount delves first into isolation and cellphone culturing then maintains with themes corresponding to phagocytosis, genetic manipulation, macrophage activation, and lipid signaling. Written within the hugely profitable Methods in Molecular Biology™ sequence structure, chapters contain short introductions to their respective topics, lists of the mandatory fabrics and reagents, step by step, quite simply reproducible protocols, and notes on troubleshooting and heading off recognized pitfalls.
Authoritative and state-of-the-art, Macrophages and Dendritic Cells: equipment and Protocols presents a well timed and important advisor for either pro investigators and neophytes pursuing this valuable box of study.
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Extra resources for Macrophages and Dendritic Cells: Methods and Protocols
6. To disaggregate clusters of BM cells, the cell suspension is passed through the syringe and filtered using a cell strainer (pores diameter = 70 μm) into a 50-mL tube. 7. Cells are recovered by centrifugation and the pellet resuspended in 10 mL R1 medium. 8. Finally, cells are counted using a Bürker chamber. 3. Preparation of Cell Suspensions from Spleen 1. Mice are euthanized using CO2 asphyxiation followed by cervical dislocation (see Note 4). 2. Spleens are isolated from mice, placed in a 60 × 15 mm petri dish containing 5 mL of IMDM complete medium, and cut into small pieces.
11. Add 40 μl of α-ISPP 6% dissolved in EtOH to all tubes including the standards. Vortex for 5 s (see Note 2). 12. Incubate all tubes at 95°C for 30 min. 13. Incubate all tubes at 4°C for 30 min. 36 Classen, Lloberas, and Celada 14. Transfer 200 μl from each tube to a new 96-well cell culture plate. 15. Read the optical density at 540 nm and convert this number using the standard curve in μg urea. 16. Analyze data using the following formula: μg urea/60 (molecular weight of urea) × 50 (dilution factor)/t (minutes of incubation with arginine) = units of arginase per 1 × 106 cells; 1 unit = amount of enzyme required to hydrolyze 1 μM arginine per minute.
In order to measure the concentration of mGM-CSF, a specific enzyme-linked immunoabsorbent assay should be performed. 2. Preparation of Cell Suspensions from Bone Marrow 1. Mice are euthanized using CO2 asphyxiation followed by cervical dislocation (see Note 4). 2. The skin of mouse hind legs is pulled back and the muscles dissected to expose femur and tibia (see Note 5). 20 Mortellaro et al. 3. Using a scissors and a pair of tweezers, the knee joint is cut, the muscle along the femur is sliced and the bone is severed from the hip joint.
Macrophages and Dendritic Cells: Methods and Protocols by Georgia Schäfer, Gordon D. Brown* (auth.), Neil E. Reiner (eds.)