I have not worked with 96 well plate; I need to split my cells into 96 well plate, seeding 0.5 cell/200 micolit media/ well. How I can plate 0.5 cell/well. Is there any formula to use? I think I need 50 cells/20ml media at 100 wells? Am I right?
I have also carried out these experiments. Half a cell per well, really means that you should get one cell every two wells. Perfect for hopefully making sure that you will be able to pick a single clone.
Resuspend your cells to be about 10 million per mL.
Then place 200 uL into the first column of a 96 well plate. Then place 150 uL in columns 2-12. Then carry over 50 uL from column 1 to 2, and so forth doing a quarter serial dilution across the plate. This will give you the following: approximately in each row.
1: 2097152
2: 524 288
3: 131072
4 32768
5: 8192
6: 2048
7: 512
8: 128
9: 32
10: 8
11: 2
12 0.5
of course you can always start with 1 million per mL, and then column one would be approximately 200K per well, and half a cell per well would be mid way through the plate. You can keep going though with the serial dilution across the remaining columns, increasing the chance of getting a single colony. It all depends on how many plates you wish to make.
Could you please be more specific? 0.5 cells is half a cell per well, that means that you could have 1 cell/0cell per well, what kind of experiment is this?
I agree with Ana. What kind of experimental design it is!!!! I am kind of confused. 0.5 cells for plating is unreasonable and before performing any experiment and asking any question I advice Hind to read and understand the experiment in a practical way. I think Hind's requirement is probably 0.5 million cells (0.5X10^6)/200ul). Calculation for diluting cells for seeding is very straight forward. You have to count the number of cells in your stock (using hemocytometer or by any other automated counter). You have to exclude the dead cell count to get the exact live cell number. Then according to your need you diltute the stock using complete sterile culture medium and mix it gently and plate in 96 well plate or else where by dispensing required volume that contains the exact number of cells you need. You have to remember different cells have an optimized seeding density/square cm area.
If you really need 0.5 million cells (0.5X10^6)/200ul) which seems to me quite high and unreasonable. Please check the exact number of cells you really need.
Thank you so much; my goal is to get a single clone. I want a density of 0.5 cell ( half a cell) per 200 ul in each well. This experiment is to Identify single clones.
I have also carried out these experiments. Half a cell per well, really means that you should get one cell every two wells. Perfect for hopefully making sure that you will be able to pick a single clone.
Resuspend your cells to be about 10 million per mL.
Then place 200 uL into the first column of a 96 well plate. Then place 150 uL in columns 2-12. Then carry over 50 uL from column 1 to 2, and so forth doing a quarter serial dilution across the plate. This will give you the following: approximately in each row.
1: 2097152
2: 524 288
3: 131072
4 32768
5: 8192
6: 2048
7: 512
8: 128
9: 32
10: 8
11: 2
12 0.5
of course you can always start with 1 million per mL, and then column one would be approximately 200K per well, and half a cell per well would be mid way through the plate. You can keep going though with the serial dilution across the remaining columns, increasing the chance of getting a single colony. It all depends on how many plates you wish to make.
If all else fails, an alternative is to seed your cells i clonacell media:
http://www.stemcell.com/en/Products/All-Products/ClonaCellTCS-Medium.aspx
This will also allow single clone isolation.
All the best.
Ad Amar Yeware: If you carefully read all postings, you will understand the question.
Ad Hind Albeshri: If you have access to a FACS single cell flow sorting might be another option.
Hi,
charles protocol is so useful and usually is used in hybridoma technology.but you can do this diluting step in test tube.after cell counting if you dilute cell suspension as many as 5 cell/well and then 2.5 cell/ml , in 200 micro-litter per well you will have 0.5 cell/well.
Thank you all, I really appreciate your help. Mehrdad that what I did, thank you again
This technique is called "limiting dilution". It is used to isolate single clones. Basically, you dilute cells sufficiently enough so that when you plate a certain volume of the cell suspension per well, you are statistically guaranteed of a few wells that contain only one cell. When one says 0.5 cells per well, it does not literally mean half a cell per well. What it means is that you hope to get one cell per 2 wells. Your pipetting technique is crucial for the success of this approach. Your cell counting technique and serial dilution technique have to be reliable. Make sure that you mix the suspension regularly so that cells don't sediment when you are aliquoting 200 ul per well. When I do it, I prepare a suspension of 1 million cells per ml (1000 cells per microliter). Either you can do a 1:10 serial dilution (best method) to obtain the desired cells per volume or take a specific volume of suspension and dilute it in an appropriate volume of complete media to obtain 0.5 cells per 200 ul. Ideally, after you plate, you want to identify and mark wells that have a single clone (assuming that you are doing single clone isolation). This can be time consuming. I look at the plates after 2-3 days and look for clonal expansion. You can easily identify individual clones. Hope this helps.
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