![]() 1: 2 Transfer 2.5 ml of suspension to a new T25 flask and add the complete supportīut if the growth support in which you want to pass your cells does NOT have the same area as the starting one, how to do and calculate the correct dilution of the cell suspension?.1: 5 Transfer 1 mL of the suspension to a new T25 flask and add the complete support.1:10 Transfer 0.5 ml of the suspension to a new T25 flask and add the complete support.If you suspend your cells post-trypsinization in 5ml of medium you can split your cell at different ratio You can resuspend your cell in, let’s say, 3ml, then put 1 ml in a new T25 flask, adding 4ml of fresh medium to the flask OR you can resuspend your cells in 15ml, then put 5ml of cell suspension (1/3 of the original) in a new flask. What if you had to split your cell with a 1: 3 split ratio? To divide the cell suspension 1: 2, you can put half the amount of cell suspension (2.5 ml) in a new T25 and add 2.5 ml of new medium (if you usually put a total of 5 ml of medium in your T25). Suppose to trypsinize the cell culture growing on a T25 flask and, after centrifugation, resuspend the cells in 5 ml of medium. = split ratio (let’s say 1/4 = 0.25, referring to the previous example) V = volume in which you resuspended the trypzinized cells X= volume of cell to be passed in the new vessel If it is therefore necessary to divide the cells in a ratio of 1: 4, it will be necessary to move 1/4 of this volume X of cell suspension (X / 4, containing 1/4 of the cells of the original suspension) to the new growth vessel aand add cell-free growth medium to reach the volume of medium in which you usually growth your cells. If it is therefore necessary to divide the cells in a ratio of 1: 2, it will be necessary to move half of this volume X of medium (X / 2, containing half the cells of the original suspension) to the new growth vessel and add cell-free growth medium to reach the volume of medium in which you usually growth your cells. you move the cells from a T75 to a T75), it is super easy.Īfter trypsinization and centrifugation to remove trypsin, the cells are resuspended in a specific X volume of medium. If the culture vessels in which you want to subcultivate the cells have the same surface area of the starting vessel(e.g. How to do in practice? splitting between two identical culture vessels Therefore, if the split ratio is 1: 2, it means that half of the cells in the initial culture need to be moved to the new subculture vessel (with surface equal to that of the starting trypzinized culture vessel) if the split ratio is 1: 3, only one third of the cells from the original culture will need to be moved to a new carrier vessel to give rise to the new culture, if 1: 4, then 1/4 of the cells and so on. The split ratio indicates which fraction of cells to subculture (or pass) into the new growth vessel. How are split ratio, population doubling (PD) and time to subculture correlated?Īt which split ratio is it best to pass the cells? ![]() Splitting between two different culture vessels Splitting between two identical culture vessels ![]() Sometimes after trypsinization for routine subcloning, when you are already familiar with the cells or as required by the datasheet (and better if you have the growth curve of your cells and know the doubling time of the cell population), you can split the cells not by countingthem but simply by diluting the cell suspension to an appropriate splitting ratio.
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