By Dieter Körholz, Wieland Kiess
Describes for the amateur investigator a number of novel applied sciences and molecular recommendations in particular designed to check mobile immunology and advertise its gene treatment applic- ations
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You may also stain just an aliquot of 100 µL or so in a smaller tube (we use Falcon 2052 tubes) and then proceed with the respective smaller volumes. 10. Add a 20-fold volume of BD lysing solution to your tube and incubate for 10 min at room temperature. 11. Spin down and remove supernatant by careful decanting or aspiration. 12. Add 1 mL of permeabilizing solution and incubate for 10 min in the dark at room temperature. 13. Complete like PBMC procedure. 2. Reagent Preparation and Storage (Selected Reagents) Brefeldin A should be dissolved in DMSO and stored in small aliquots at high concentration at –80°C.
2001) Review. Modulating the immune response with dendritic cells and their growth factors. Trends Immunol. 22, 41–47. 20. , and Nishimura, T. (1998) Functional skewing of bone marrow-derived dendritic cells by Th1- or Th2-inducing cytokines. Immunol. Lett. 67, 63–68. 21. , et al. (1996) Cytokine expression by human peripheral blood dendritic cells stimulated in vitro with HIV-1 and herpes simplex virus. J. Immunol. 157, 4028–4036. 22. , et al. (1999) Survival, maturation, and function of CD11c– and CD11c+ peripheral blood dendritic cells are differentially regulated by cytokines.
15. Incubate for 30 min at RT in the dark. 16. Add 2 mL wash buffer and vortex gently. Flow-Cytometric Immune Function 47 17. Centrifuge 10 min at 500g. 18. Decant the supernatant. 19. Resuspend the now intracellular-stained and permeabilized cells in approx 200 µL wash buffer. 20. Acquire the samples in )1 h on the ﬂow cytometer. 21. Store samples at 4°C if not acquired immediately (see Note 2). 2. Sample Processing: Functional Surface Antigen Detection Sample processing for functional surface antigen detection includes cell surface staining with ﬂuorophore-conjugated monoclonal antibodies followed by red blood cell lysis.