In: Respiratory18 Jul 2014
Discontinuous gradients of metrizamide (Nycomed Pharma AS; Oslo, Norway) were prepared from 27% metrizamide stock solution diluted with 10 mM HEPES-buffered saline solution at a pH of 7.3 containing 0.1% each of glucose and gelatin. The method was derived from Fukuda et al, and Vadas et al, in which metrizamide-containing solutions are isotonic. Up to 107 BAL cells were resuspended in 2 mL of HEPES-saline solution and layered on top of increasing concentrations of metrizamide gradients (3 mL at 27%; and 2 mL each at 22%, 20%, and 18% metrizamide) in 15-mL transparent polypropylene centrifugation tubes (Corning; Corning, NY). After centrifugation at 1,200 X g for 45 min at 18°C, the cells were collected from each interface, washed with Hanks’ balanced salt solution containing 2% FBS and were counted before being resuspended in MEM supplemented with 5% FBS. http://help-in-mens-health-problems.net/ fully The total cell recovery after density gradient centrifugation ranged from 70 to 75%.
Preparation of AM Subpopulations
Results of preliminary experiments, in which BAL cells were fractionated on several layers of metrizamide (16%, 18%, 20%, 22%, and 27%), indicated that > 95% of total AMs recovered after centrifugation localized to the interfaces of gradients <22% metrizamide. The 22% metrizamide cutoff value was based on the distribution of blood monocytes that were used as a reference for buoyant density (see below). The cells recovered from the top of the 16% metrizamide layer had a similar morphology’ to those recovered from the 16 to 18% layer, and these were therefore combined in subsequent experiments. Overall, we prepared three subpopulations of AMs from interfaces as follows: < 18%; 18 to 20%; 20 to 22% metrizamide. The macrophage phenotype of cells in the three isolated populations was confirmed by the alkaline phosphatase antialkaline phosphatase method, by using MR-1 monoclonal antibody (Serotec Ltd; Mississauga, ON, Canada) that recognizes a cytoplasmic component of guinea pig macrophages and monocytes (Fig 1). Using the same centrifugation procedure as for BAL cells, blood monocytes predominantly localized to the 20 to 22% metrizamide interface and were not present in the lower-density gradients.
Figure 1. Immunostaining with MR-1 antibody (red color), confirming the macrophage phenotype of isolated hypodense (top left, A), intermediate-density (top right, B), and liigh-density AMs (bottom left, C). Bottom right, D: unfractionated AMs stained with isotype-matched, nonspecific primary antibody (alkaline phosphatase-antialkaline phosphatase reaction; Mayer’s hemalun counterstaining; Scale bar represents 50 μm).
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