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As an alternative to using unpurified OPN^+^ OS cells, we were able to enrich for the OPN-expressing cells, by first generating a density gradient using Percoll. Next, we used a modified fluorescence activated cell sorting protocol which included a suspension step in order to enrich for primary human OS cells and to improve OPN immunofluorescence signal (magnification x1000).

After the enrichment process, a range of primary cell numbers were verified with fluorescence microscopy and flow cytometry. Normal human bone marrow cells were used as a positive control for OPN immunostaining. As a negative control, no OPN staining was detected in the negative control sample, when used at the same fluorescent settings to the OPN^+^ sample (Figure 7{ref-type=\"fig\"}). These results showed that by using a density gradient and cell enrichment techniques, it was possible to specifically identify and isolate OPN^+^ OS cells from their surrounding bone marrow fraction.

![OPN is expressed in primary OS cells.
Normal human bone marrow (BM) cells were used as a positive control for OPN immunostaining. No staining was detected in the negative control sample, when used at the same fluorescent settings to the OPN^+^ sample.](pone.0076234.g007){#pone-0076234-g007}

It is thought that OS cells have a long half-life in vivo and that OPN may be involved in the recruitment of other cells types, including inflammatory cells, to the site of a developing bone lesion [@pone.0076234-Sternheim1]. As OS cells are likely to have longer half-lives in vivo, the enrichment technique presented here could be used with the aim of differentiating primary OS cells, from\nhttps://documenter.getpostman.com/view/21913293/UzkcRsWE https://documenter.getpostman.com/view/21884699/UzkcRsWC https://documenter.getpostman.com/view/21830230/UzkcRsWF https://documenter.getpostman.com/view/21888411/UzkcRsWD https://documenter.getpostman.com/view/21831849/UzkcRsWB # Introduction\nWhat does your API do?

Overview

Things that the developers should know about

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What is the preferred way of using the API?

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Need For Speed Movie Dual Audio 720p 29

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