Download Advanced Methods in Cellular Immunology by Rafael Fernandez-Botran PDF

By Rafael Fernandez-Botran

Immunologists to boot investigators in different disciplines could frequently use protocols regarding the isolation, cultures and characterization of alternative varieties of leukocytes. complicated equipment in mobile Immunology is a suite of strategies in an easy-to-use format.Each bankruptcy offers readers with comparable software info, a step by step description of the technique, substitute thoughts, pertinent references, and data approximately advertisement assets for fabrics and regents. as well as leukocytes, the authors advisor readers during the methods of telephone tradition in addition to irritation and autoimmunity in quite a few animal models.Covering themes reminiscent of PCR and Apoptosis, this e-book will serve a advisor to commonplace techniques in mobile immunology whereas using either human and murine versions.

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4 Materials and reagents • Eosinophil preparation (as described) • HUVEC (ATCC) cultures • Collagen (calf skin, type I, Sigma C-9791). 50 µg/ml solution in acidified water. 0 • 96-well tissue culture plates • Incubator (37ºC) • Pipettes • Multi-well spectrophotometer Protocol 1. Prepare 96-well plates by coating with collagen or HUVEC monolayers. For collagen, coat wells by incubating with 30 µl/well of collagen solution for 30 min at 37ºC. For HUVEC monolayers, culture HUVEC cells (2 × 105/ml) in collagen-coated wells for 24 to 48 h before the adherence assay.

7. 5 to 10 µM H2O2, final) instead of the neutrophil suspension. Protocol (kinetic method) 1. First, create standard H2O2 curve. 5 to 10 µM). 2125/ch02 Page 39 Thursday, September 6, 2007 3:37 PM Chapter two: Isolation and characterization of neutrophils 39 2. Incubate for 5 min at 37ºC. Determine the fluorescence of each standard and construct standard curve. 3. 2 ml of the neutrophil suspension in a clean cuvette. 4. Place cuvette in spectrofluorometer and begin stirring. After allowing approximately 2 min for the temperature to equilibrate (37ºC), record baseline fluorescence.

5 ml of saline to each tube. 4 ml saline. 11. Remove 1 ml aliquots from each tube and transfer to scintillation vials. Add 10 ml of scintillation fluid to each vial. Count in scintillation counter. 12. Calculate the percent phagocytosis according to the formula: % phagocytosis = (sample cpm) × 100 ÷ (control cpm) Comments The labeled bacteria suspension is prepared by growing S. aureus 502A (ATCC) in Trypticase soy broth (TSB) containing 10 µCi/ml of a 14C-labeled amino acid or uracil for approximately 4 h at 37 ºC in 2125/ch02 Page 34 Thursday, September 6, 2007 3:37 PM 34 Advanced methods in cellular immunology a shaking incubator.

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