Hi,

I suppose that your problem is that EDTA is difficult to dissolve, isn't it? If not, please tell me.

Add the appropriate quantity of EDTA to PBS and keep it stirring. You can heat it moderately to promote its solubility.

Keep all this until the solution becomes transparent.

I hope my advice helps you

The pH needs to be 8.0 to get the EDTA to dissolve. You can make an EDTA solution in water and add it to PBS to the correct dilution and correct the pH if necessary. Also use PBS without calcium and magnesium. 

http://cshprotocols.cshlp.org/content/2009/12/pdb.rec12064.full?text_only=true

If you handle blood cells, as monocytes, be sure that final pH is 7.4 as pH of blood, otherwise cells could be damaged. EDTA can dissolve at this pH with stirring and heating moderately.

The proposed protocol is a stop solution with detergent Tween20 not intended for living cells, but if you need PBS with EDTA frequently, it could be a good idea to prepare a concentrated stock solution of EDTA in water to be added to PBS as Lisa propose but in my case I don't need to change the pH to dissolve it. In any case, final pH must be 7.4.

Finally, to remove adherent monocytes, PBS without calcium is not enough because it doesn't remove existing calcium of existing adherent interactions. A chelator, as EDTA, is needed to sequestrate existing calcium and to break the adherent interaction.

Thank you Lisa and Mario for your kind help. Please, can you detail the precise preparation procedure from EDTA (powder) and 1x PBS (solution)?

As we commented, you have 2 ways to do it.

1. You can dissolve directly (with stirring and moderate heating) the EDTA in your PBS solution (I suppose your solution is at pH 7,4). It's a percentage so the quantity of EDTA depends on the volume of your PBS solution. For example, if you want to prepare 1 L. you'll need  (1000x0.02)/100 = 0.2 grams. Probably, in spite of being a buffered solution, your pH could vary, so in the end, check the pH and correct it if needed.

2. You can have a concentrated stock solution of EDTA, for instance 15% so you'll need 15 g for 100 ml of water. In this case, water is not a buffered solution so pH will decrease until 4 and in this case you have to add NaOH for increasing pH to 7,4 or 8. When you have prepared your stock EDTA solution, the next step is to calculate the volume of this stock solution needed in your PBS solution. If the final concentration of EDTA is 0.02% and if you want to prepare 1 L of PBS+EDTA, you'll need to add 1,333 ml of EDTA stock solution to 1 L of PBS solution. In this case you dilute very slightly the components of PBS solution but it's very slight so it's not a problem and if you have to prepare this solution often, it could be a faster way (after preparing stock EDTA solution). As usual, check if final pH is 7.4

Many thanks, Mario!

Can someone please suggest a protocol/step by step instructions how you use the PBS-EDTA solution to dissociate cells while splitting, as an alternative to Trypsin? I have PBS without Calcium and magnesium. I also have a stock of 0.5 M EDTA solution stock that I found in the lab. What is the advised concentration to use for dissociating adherent cells? I assume I need to mix it with PBS-/-. What do you pH adjust to, before adding to your flask/dish? Thanks