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Cell Biology Lab 4 Trypan blue staining and cell counting

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Cell Biology Lab 4 Trypan blue staining and cell counting

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    1. Cell Biology Lab 4 Trypan blue staining and cell counting PCB 4023 L TA: Sushmita Mustafi

    2. Objectives: What is cell counting? Why do we need to count cells? What is hemocytometer? How to handle hemocytometer? What is trypan blue? What is dilution factor? How to calculate cells? How to perform the procedure?

    4. Identify a hemocytometer from regular glass slides

    5. Cont.

    6. Trypan blue live cell membrane is impermeable to trypan blue, but dead cell membrane absorbs trypan blue. Hence trypan blue is used in cell viability test.

    7. Dilution of cells. In order to count cells Detach cells from the flask. Pipette and mix well to remove clumped cells. Collect small amount [ 100 L to 500 L] in eppi tube. Add trypan blue into the cells in eppitube. Depending on the amount of trypan blue calculate the dilution factor. Dilution factor = Final volume / initial volume. Ex. 100 L of cells and 100 L of trypan blue DF= 200 L / 100 L = 2

    8. Counting grids of hemocytometer

    10. To prepare the counting chamber The mirror-like polished surface is carefully cleaned with lens paper. The cover slip is also cleaned. The cover slip is placed over the counting surface prior to putting on the cell suspension. counting chamber is then placed on the microscope stage and the counting grid is brought into focus at low power. One entire grid on standard hemocytometer can be seen at 40x Clean the cover slip and the mirror like surface after use. Never scratch the mirror like surface of hemocytometer while cleaning.

    11. It is important not to overload the chamber, as doing so will give an inaccurate count. The same is true if the cover slip is moved after the sample is loaded.It is important not to overload the chamber, as doing so will give an inaccurate count. The same is true if the cover slip is moved after the sample is loaded.

    14. Trypan blue, which is carcinogenic so be careful! Viable (living) cells will not take up the dye, where dead cells will (see figure 5 above). Dead (i.e. stained) cells should be excluded from the count. Make sure not to leave your cells exposed to trypan blue for a prolonged period of time; eventually even living cells will absorb it and appear non-viable. Trypan blue, which is carcinogenic so be careful! Viable (living) cells will not take up the dye, where dead cells will (see figure 5 above). Dead (i.e. stained) cells should be excluded from the count. Make sure not to leave your cells exposed to trypan blue for a prolonged period of time; eventually even living cells will absorb it and appear non-viable.

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