Hi all,
I'm working on differentiating ESCs to a forebrain lineage and was wondering how to calculate differentiation efficiencies? I see efficiencies written in some ESC induction papers (example, some papers quote a 20% differentiation efficiency from their ESCs) but am not sure how these are calculated.
Are they from calculating the number of cells in the final plate (at the end of the differentiation time line) that are positive for a lineage marker vs. the number that aren't?
Example: Final plate is stained with an antibody against a lineage specific protein. 20% of the cells in the field of view (based on DAPI) is positive for this marker. So efficiency is 20%?
Or is it based on number of starting cells divided by number of cells in the final culture positive for a lineage specific marker?
Example: Start with 10000 ESCs dissociated and plated. Count number of cells throughout differentiation protocol (assuming that cells are dividing at least initially after plating out, lets say cell number plateaus at 1 million). After differentiation protocol, end with 10000 cells that are staining positive for the lineage marker.
Would the efficiency then be 10000 (# marker positive cells) /1000000 (cell number at plateau) = 0.01, or 1%?
Or is there some other (probably smarter) way of calculating differentiation efficiency?
I've seen either being done. They should say how they calculated it in the paper.
If there's cell division going on during differentiation, then obviously the second number is going to look better. So that can be used to "trick" an audience that's looking only superficially into overrating the result.
I think the answer lies it self in the query..
Make a time line for emergence of fore brain markers in the culture, which will highlight their exact days for the stages specific expression. They using flow cytometry you can calculate the percentage in appearance and disappearance of markers. You can validate by q-PCr also.
As already mentioned, it should be mentioned in the paper how they have calculated the percentages of their differentiation efficiency. Anyway these kind of quantifications are mostly performed on the basis of positive cells compared to all cells in your analysis (it can be e.g. flow cytometry or IHC/IF). Especially, if a cell population in a transition step should be analyzed you´ll have always the problem that some cell hadn´t yet reached this point but will and some cells are already further differentiated which shows that the right time point will maximize your number of positive cells and thus your differentiation efficiency.
From my personal experience I can only mention that these calculated differentiation efficiencies often vary if a published protocol is adapted to a different lab.
Hello Amy,
You can't count around 10,000 cells positive for a specific marker under a microscope and also you are going to compromise your accuracy by same. To be very specific and brief, you can calculate the percentage positivity for a marker which is positive for that partiular lineage by FACS.. Percentage positivity gives you a direct indication of differentiation efficieny. Suppose at control level ESCs are expressing MAP-2 which is a mature neuronal marker, as 0.5% and after differentiation protocol its 95.5% then obviously the efficiency of ESCs to differentiate into mature neuron is 95%. Through FACS you can get a direct record of postive percentage and no need to go through complicated formulas or equations...
Last but not the least, comparision of control and differentiated population is must to get final accurate value of percentage efficiency and your marker of choice should be highly specific for that lineage.
Hope it helps..!
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