Try DNAse

I also met this problem. You'd better to resuspend your sample with medium (without FBS). I think FBS make the plugs.

You may be using too much antibody - which will aggregate cells - or you may be overloading your column with too many input cells.

My guess is you have too many labeled cells on the column. Estimate the number of labeled cells (~90-95% of PBMCs will not be naive CD4) and look up the maximum number of labeled cells you can use for each column (on datasheet). Use a bigger column if possible or you can split the sample over 2 columns if it is too much.

Thank you for all your help. But I don't think too many labeled cells causes this problem. Because when I use the column to positive select CD4+ T cell from a spleen. The column still plugs in this condition. 

And I have asked the question to the technician in miltenyi company. He says it  may be caused by the bubble in the buffer. When I load my sample to the column, I also load the bubble, maybe this is the cause of this problem. Next time I will carefully  remove the bubble to see if this happens again.

Dear Yang,

The bubble are exactly the reason for clogging. We had to struggle a lot for this too. My advice is that you must degas the elution buffer correctly. Use a vacuum pump if necessary for 5 mins. 

Another observation that I have seen is that, suppose you have taken 1 ml of solution to apply in the column do not pass the the full 1 ml, rather pass 900 ul or so. As, if you apply the full solution then you will inject air from the top of of the solution in  the tip of the micro pippett in the column too.

Hope this helps. Happy sorting.

Shibabrata

Miltenyi also recommend including EDTA at 2mM in the MACS running buffer, which may help to reduce cell-cell adhesion and thus reduce clogging.  

If you don't have air bubbles in the column, your sample is free of particulate debris and you have included EDTA the problem is likely to be too many cells. The column can definitely clog with too many cells whether labelled or not, so in this case you should divide the sample over 2 columns as was previously suggested.

@Gamage A M , Before each loading, I do have used the nylon filter to filter the sample, even the washing buffer. But it still clogs. So I think it is the bubble in the liquid takes the main responsibility for this problem.

I have done the experiment again and found that FBS was the reason for column clogging. I changed the serum containing buffer to PBS and the clogging was solved.

And thank you for all your help!

Thank you for letting us know that it was the use of FBS that clogged the column.  

Why did you use FBS rather than the Miltenyi "Wash buffer", which is 0.5% BSA/2mM EDTA/PBS-Ca/Mg-free?  (I make my own buffer and leave out the 0.05% azide to keep my cells fully functional - use it under sterile conditions - store it in the refrigerator) The directions mention that buffers containing Mg++ or Ca++ are not recommended.  FBS is known to have variable concentrations of these ions. If concentrations were too high in your particular batch of FBS - it would allow cells to adhere to each other (through Ca/Mg-dependent adhesion molecules) -  EDTA is required to chelate these ions and prevent inter-cell adhesion- which will clog the column.  

@Laura Timares

I make my wash buffer with 1% FBS/2mM EDTA and PBS. Because I don't have BSA, so I instead BSA of  FBS. And FBS will make cells survive better during the process of cell isolation.

I have not had any problems with the column blocking with 2%FBS, 2mM EDTA in PBS.  Are you sure your cell density wasn't too high?

@Stephen Juvet

Yes, I am sure. I loaded total cells(1X10^8）in 1ml buffer to 25 LS column.  And it was clogged. But when I changed the wash buffer with PBS, the clogging problem didn't exist. So I think FBS is the reason for clogging.  

Did you filter your FBS before use? FBS can sometimes be contaminated with insoluble material. Maybe it would help if you remove it before adding the FBS to your buffer.

I have filtered FBS before use

It depends on your starting product.

PBMC's or PBSC's.

PBSC's will annoy the heck out of you if you try to use the columns without running 1-2 Ficolls.

You might want to run ficoll 2 times if you don't have dnase. Dead granulocyte clumps are less dense than just plain dead cells, so they'll act as barriers preventing the actual granulocytes from going to the bottom.

Or just keep filtering more than you've done.

Also, make sure that you're basing how much you're adding to the miltenyi collumn on TOTAL CELLS! NOT your target cells.