Contando células-tronco neurais

Now I'm going to show you how to count human neuro precursor cells and you would count your cells when you want to put them for immuno chemistry or when you want to do a transfection with nuclear factor machine. I use a phase contrast hemo cytometer that has a, an edge grid and also we use a dye called trip and blue. And the dye is useful in that it can differentiate between live and dead cells.

Dead or dying cells take up this dye and they appear blue. When you look at the cells in the hemo, cytometer and live cells are able to exclude this dye and they, they, they don't appear blue. So that way you can determine the viability of your cells to prepare a solution of trip and blue and media.

I'm going to add 20 microliters of trap and blue solution into this 0.5 mil epi tube. And then I'm going to add 25 microliters of media. So that way right now I have 45 microliters of, of a trap pan blue and media solution so that when I add five microliters of cell suspension, I'm going to have a one to 10 dilution.

And so now I'm, I'm going to add five microliters of cell suspension. First I'm going to make sure cells are well resuspended by finger vortexing. So now I want to make sure this solution is well mixed.

So I'm gonna put bed up and down a few times. And finally I'm going to load about about 12 microliters of this cell suspension into a hemo cytometer. It has, it has two ports and you can, you can use either one.

So all you have to do is introduce the solution at this port and the liquid is gonna be sucked up by a capillary action. And now I'm ready to count. Here is a look at at the hemo cytometer at 10 x and here we have a quadrant which contains 16 squares.

And as you can see, we can observe here live and dead cells. Here we have live cells such as this and this and this. And we also have some dead cells such as this one.

If you notice, the dead cell appears dark blue, whereas live cells have sort of a hail around them. So now I'm going to count all the live cells. So I'm going to go from left to right and top to bottom.

1, 2, 3, 4, 5, 6, 33, 1 32, 33, 34, 3 5, 3 6. So we have three six cells total in this quadrant. So when I see cells that are on the borderline of the quadrant, such as along this line right here, to be consistent, I count cells that are on the left border and on the top border.

And I consistently count cells on those borders for all the quadrants on the hemo cytometer. So now we are going to calculate how many cells we have per microliter. And in order to do that I'm going to take the average of the cells per quadrant, which is 38 and multiply by 10, which is a factor determined by the dimensions of the chamber.

And we're going also to multiply by 10, which is our dilution factor in this case. And in the end, we're going to get the number of cells per microliter. So in this case we have 3, 800 cells per microliter.

And since we resus suspended our cell pallet in 500 microliters of solution, you can calculate how many cells total you have. And we are all set.