Hi Aimee,

The Trucount beads are essentially just a flow cytometry tube with a specified number of small beads in it (around 50.000 but varies slightly between batches). The beads were developed for staining of lymphocytes in whole blood to be able to determine the concentration (cells/ml) of stained cells in the samples.

Any flow cytometry staining that works without Trucount beads should work with them, with one important caveat - the beads are only guaranteed to work in lyse-no-wash protocols (i.e. you should not have any washing steps after adding your sample to the beads, i.e. just dilution/red cell lysis added before analysis) since it can be hard to guarantee the number of cells and beads that are lost during a wash step. In principle you could even stain your cells first, spin them down and re suspend them in a known volume and then add that to the beads (although I have never done this myself).

The other important point is that you need to add a highly accurate volume of sample (i.e. blood or sample in solution; for blood often 50µl). The reason for this is that you then know the number of beads that corresponds to a certain volume of cells (if using 50 µl of blood then approximately [depending on batch; se above] 1000 beads per µl of blood). Any other reagents added are not relevant since this ratio will be the same - 1 µl of blood equals 1000 beads.

When the sample is run on the flow cytometer you will then be able to find the beads in any channel (this is possible in SSC/FCS or [usually better] in a fluorescent channel were they are bright). When you have collected enough events of the cell population that you are interested in you will then be able to determine the number of events you have of that cell type, but also the number of events of beads that were analyzed during the experiment. The number of beads will then tell you the volume that you have analyzed of your original sample since a certain number of beads corresponds to a volume (i.e. 1000 bead events correspond to 1 µl of sample in the blood example above). Based on that calculation you can determine the total number of cells in your original sample by multiplying calculated concentration of cells to the total volume of the original cell sample (i.e. before removing a small sample for the trucount analysis).

To answer your questions above.

i) You do not need any other controls than you typically use for your samples

ii) The amount of antibody is not relevant. The 20 µl is (i pressure) based on using the TBNK mix that BD supplies to determine the concentration of CD4+ and CD8+ T cells, CD19+ B cells and CD16/56 + NK cells in blood.

thank you!

Mats gave a perfect answer and even answered the question I had about which volume to use for the calculation, i.e. is it the 50 uL of sample or the total volume added including the antibodies or lyse solution. Thank you Mats!

Hi everybody, does anyone knows the size of the beads in the trucounts?

thnks!