I've done quite a lot in just over a week. After learning how to culture human microvascular cells, I've now got two large flasks of them growing nicely in the incubator!
Today, I've been immunophenotyping one set of the endothelial cell lines with anti-CD31 antibody. This is to identify that this population of cells are of endothelial cell origin (CD31 is expressed in endothelial cells). In its simplest, the process involved removing the cells from the culture flask (using dissociation media); centrifuging them into a precipitate, resuspending and then pipetting 250,000 of these cells into each of the eight tubes. These were then incubated with primary antibody (CD31). As this antibody wasn't conjugated with a fluorescent marker, a secondary antibody (FITC) was used. Incubation for each antibody was for 20-minutes at 4-degrees. So, at the end of this process, I had 8 tubes: 2 unstained, 2 with secondary antibody only, 2 with isotype and secondary (negative control) and 2 with both primary and secondary staining. These samples were then captured using fluoresecent-assisted cell sorting (FACS) to show CD31 expression in this cell population.
Tomorrow, I need to check on my growing cells, and next week, repeat the process above but on another type of endothelial cell.
So, you now know what I've been up to in the lab! In the evenings,
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