There is no specific brand we use. We have use commercial products and made it up from powder. With either version, watch out for particles in the trypan blue. I find they will always appear given enough time. These particles can through off the reading from the Countess.
Thank you for your informative video 🙂 I would like to ask for a clarification: I assume that once the live cells are calculated, one needs to define how many cells there are in the volume they have for downstream steps. For example, if you had resuspended your cells in 200μl of medium and you used, let's say, 10μl for the measurement it means that the number of cells per ml needs to be calculated afterwards on the 200μl basis (Because this is what you have, not an ml) and also you need to subtract 5% of that, which is what you used for the measurement, right?
The final units you will be in cells/mL. To determine the number of cells in a particular volume, use the cells/mL as your conversion factor. For example, if you used 10 uL of a 2000 cells/mL solution (10 uL = 0.010 mL) the calculation would be 0.010 mL * 2000 cells / 1 mL = 20 cells (mL cancel out). The concentration does not change as you remove volume from the sample (as long as it is homogenous or fully mixed). For every volume you take, you would multiply by the same concentration to get the number of cells. Hope this helps.
Hi Key. I recommend to you reach out to Thermofisher for suggestions on the setting for each cell line. You must adjust the gating for different types of cells. You can adjust for cell size, roundness & brightness. When I first starting using the Countess, I did reach out to Thermofisher. Unfortunately, they were not very helpful. To anyone at Thermofisher, if you are reading this, it would be great if you could recommend gating settings for some of the most popular cell lines. What I did to optimize my cell counts was to count cells on both a hemocytometer and a Coulter Counter. I then took this concentration at used it to setup the gating for the cells using the exact same sample, having the same concentration. Best I can do just now as I am on my summer vacation.
Hi Tumelo. I believe it came with the machine when we purchased it. I just checked the ThermoFisher website and this appears to be the information you are looking for. Countess™ II FL Reusable Slide Holder Catalog number: AMEP4746. Take note that this was for the Countess II. There is a Countess 3 now and I am not sure if the reusable slide and adapter are compatible. I have not had the opportunity to try it out just yet. Hope this helps.
Hello! I have two questions Can I omit trypan blue when using a cell counter? And is the dilution factor of trypan blue automatically multiplied by the cell counter? Thank you so much
I suppose you count not add trypan blue. In this case you would only get the total number of cells and would not know the division better live an dead. The Countess does all the calculations for you, including multiplying by 2 to account for the 1/2 dilution. I think there is a feature to disable the x2 function. The screen will read "Trypan Blue Corrected" if it has x2. Alternatively, you can just divide by 2 to get the cell concentration without trypan blue. Hope this helps.
Concentration is irrelevant to the volume of your solution. The volume is only important if you are trying to determine the amount of the solute in your solution. As an example, you could have 0.5 mL of a 1000 cell/mL sample. It does not matter that it is only 0.5 mL, the concentration will still be 1000 cells/mL. As the concentration is a fraction, it can be used as a conversion factor to determine how many cells are in your 0.5 mL sample. Mathematically, it would be 0.5 mL X 1000 cells/mL. The mL units cancel leaving you with 0.5 x 1000 cells = 500 cells. Therefore you have a 0.5 mL sample, with 500 cells in it, with a concentration of 1000 cells/mL. Hope this helps.
Your gating protocol please. I have started setting my own gating protocol and have been comparing results to our manual counting method. I have no other means for comparison, and I contacted ThermoFisher but they didn't provide much support. Thank you. @@ProfessorDrewCollop
I had the same issue with ThermoFisher. I contacted them to get their gating protocol, but they offered no support. I really don't understand why they could not hired someone to go through the most popular cell lines and create protocols. Here is the protocol I am using for BHK. Not sure if it is perfect, but it works for my needs. I optimized it using a Coulter Counter and a manual hemocytometer to compare. Within the Countess appropriate range, it seems give approximately the same count numbers.
Sorry, the image would not add so here it is typed out. Count setting both checked for Auto FL Threshold and Auto Lighting Size Gating = 12 - 28 Contrast Gating = 0 - 255 Roundness Gating = 0 - 75 Let me know how it works for you. Good luck.
Hi there, with regards to the total amount of cells/ml, if I wanted to determine how many cells are in my cell suspension, would I multiply it by 5 if I resuspended my cells in 5ml?
That is correct. Any fraction can be used as a conversion factor. If you want to convert mL to cells you need to multiply by a fraction with cells in the numerator and mL in the denominator. mL x cells/mL = cells as the mL cancel out.
No, you cannot use a standard hemocytometer. You can buy disposable slides, but that seems like such a waste to me. My lab has 2 of the Countess Reusable Slides. They were about $500 a piece. Here is a link to the Thermofisher site if you are interested. www.thermofisher.com/order/catalog/product/A25750
Hi Catherine. Unfortunately, I do not know the answer to that one. I have seen in the thread posted that some have been successful, while others have not. www.researchgate.net/post/Has-anyone-had-experience-using-the-Thermo-Fischer-Scientific-Countess-II-FL-Automated-Cell-Counter-for-microalgae
