Hello
I am trying to do flowcytometry for BAL fluid so i inject1ml of PBS into the trachrea and aspirate back(this done three times) . to calculate the cell count in bal i use a cell counter which gives me the number of cells /ml and a viability %. on facs machine i gate out the debris below 50K on FSC-A and then use anti cd45 antibody to gate the lekocytes.
and i calculate the total lekocytic count using this formula:
toal leukocytes= cell count/ml* vol. of BAL fluid collected * viability/100 * percentage of non debris gate/100* percentage of cd45+/100
my question is is this formula correct ???and do i gate out the dead cells twice , once in the viability and once in the non debris gate.
unfortunately i am not using a viability dye in my panel to calculate it directly from the facs machine,
my other problem i have alot of debris in my samples (sometimes 40- 50%) and the total cell count is low around 50000 cells. so does any one have advice about the ways for collecting the BAL fluid
Dear Noran Abdel latif Abdel rahman,
just some things to concider: The formula seams to be correct, but you have to take in account how your counting machine works. As an example: If your counting machine automatically already excludes debris you would make an mistake by adding the percentages of your debris measured by facs back in your formula.
Mybe just use your SSC/FSC Gate as 100 %, but that s just my take on your problem.
The amount of cells seems reasonable, at least in my hand (depending on the model) I find similar cell numbers. In highly allergic animals i tend to find more, in healthy uninflamed lungs i usually end up in about the same range.
Make sure to use cold, protein containing buffer and don't store cells.
Hope this helps.
If there are any other questions do not hesitate to ask
Best
Robert
thank you so much Robert for your answer. and for the advice i sure try to do that.
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