By Roberto Botelho
This quantity information experimental ways used to enquire phagocytosis and phagosome maturation. Chapters current equipment and protocols on quantifying uptake and phagosome maturation utilizing biophysical and biochemical techniques, proteomics, microscopy, and stream cytometry. Written within the hugely winning Methods in Molecular Biology series structure, chapters contain introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, simply reproducible laboratory protocols, and tips about troubleshooting and warding off recognized pitfalls.
Authoritative and state of the art, Phagocytosis and Phagosomes: equipment and Protocols goals to be a massive source for either specialists within the box and for these investigators delving into phagocytosis and phagosome maturation for the 1st time.
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Extra resources for Phagocytosis and Phagosomes: Methods and Protocols
Cell scrapers. 7. Glass coverslips (No. 1 thickness). 8. 6-well tissue culture plate (see Note 2). 9. Plasmid DNA. 10. FuGene HD transfection reagent. 11. siRNA: nontargeting siRNA as control and siRNA against gene of interest. 12. Neon Transfection System: System includes Neon transfection device, Neon pipette, and Neon pipette station; store at room temperature; Thermo Scientific, Waltham, MA, USA. 13. Neon Transfection System 100 μL kit: Kits includes resuspension buffer R, electrolytic buffer E2, Neon electroporation tube, and Neon tips; store all buffers at 4 °C, store tubes and tips at room temperature; Thermo Scientific.
Pellet cells at 4000 × g for 5 min and resuspend in 100 μL of PBS. Repeat once (see Note 19). 3 Opsonization of Sheep RBCs with IgG 1. Mix 25 μL of RBC with 1 mL of PBS. 2. Pellet RBCs at 500 × g for 1 min and resuspend in 50 μL of PBS. Repeat once. 3. Add 1 μL of rabbit anti-sheep RBC antibody to 50 μL of washed RBCs and rotate for 30–60 min at room temperature. 4. Pellet RBCs at 500 × g for 1 min and resuspend in 50 μL of PBS. Repeat once (see Note 19). 3 Phagocytosis and Inside-Out Staining of Phago cytosed Particles 1.
Fig. 1 Annexin V staining of apoptotic and non-apoptotic Jurkat cells and neutrophils. (a) Annexin V staining of Jurkat T cells following 4 h treatment with 1 μM staurosporine. (b) Annexin V staining of human neutrophils following 1 h heat shock at 43 °C followed by a 3 h recovery period at 37 °C. UT untreated, St. Staurosporine, HS Heat-Shock. Images are representative of 30 cells per condition, captured in two independent experiments. Apoptotic cells are stained with FITC-conjugated Annexin V, scale bars are 5 μm 34 Amanda L.
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