In my lab we only use kits from Stem Cell Technologies - they are far and away better than others on the market. I'd also do the separations manually rather than by an "auto" system, but I'm old….

Seriously tho, your should be able to easily get 95% + of your harvested cells to be the ones you want.

Often, column based techniques are sensitive to overloading (and I'm not familiar with the DynaBead products), so I'd check this, and the washing, before you invest in new kits, magnets, columns and so on.

But, as I said, we use Stem Cell.

Good Luck

G

We use column-based untouched MACS separation kits from Miltenyi Biotec for mouse CD4+ T cells and mouse CD8+ T cells (95% or better purity). We also manually sort, rather than use automation. I didn't like Dynabeads 10 years ago and have used Miltenyi ever since. Dynabeads should work, though. As John mentioned, prior surface staining could definitely interfere with bead labeling. Also Dynabeads has a variety of magnets that are not interchangeable. The magnet, kit and tube have to matchup.

Dear Adam and John

I isolated PBMCs and then used the Dynabead!

Thanks for your suggestions. I am going to try other kits as u mentioned.

Dear Dominic

Thats really a good advice. I am going to contact them and try. Thank you.

ok... but be careful that you understand the possible consequances of binding T-cells with an anti-CD3 (small beads, lack of crosslinking etc etc notwithsanding).

Good Luck

G

Use the negative purification kit from Stemcell Technologies. I have had several successfull isolations using it. The yield is not very high but purity was consistently above 94%. You can also avoid the crosslinking induced T cell activation in the process. Best of luck.

Definitely try the kits from StemCell they work really. The newest kits only take 15 mins when done by hand. I have been using these kits for 10 years with really good results in terms of yield and purity. 

Dear all

I have already tried CD8 isolation kit (negative selection) from stem cell technology and Mylteni. Unfortunately, I had still all kinds of cells (except CD4 T cells) at the end, only reduction in the percentage. I isolated CD8 from PMBCs. Is there any way to improve the isolation method? I really need the pure population. Is there anyway to combine it with other method to improve purity?

I am looking forward to your practical answers.Thank you.

If you really need highly pure population, the best way I know how is to use FACS sort if you have access to the machine. Your yield will not be very high, but you can get close to 99% purity. What are you going to do with the cells once you isolate them? As Grant mentioned, there's consequences on using anti-CD3 on T-cells...

Thanks for your comment. I have already tried sorting twice, but as you mentioned the yield was quite low. I left them in UC(HUS) +IL-2/7 +PBMCs (Irradiated)+EBV. but only 1 of them was grown(out of 400 CD8 Tcells).I added EBV and IL-7 based on pepares.Do you have any suggestions for me?I am looking forward to your comment.

Stella, I am working in allergy research , so finally I am going to ad APC+Allergens to CD8Tcells.

I use dynabeads for CD4 negative selection which works well. Never had a problem with contamination. I have tried Miltenyi system too, however I find that the dynabead is more reliable and cheaper. If you are getting impurities and you are using a positive enrichment then try to work on ice if you are working with negative enrichment then I would suggest to leave the antibody and bead incubation for longer. 

I don't know if you're working with human or mouse cells, but in my opinion you should only use negative-selection strategies if you want to do functional studies with the cells. A relatively cheap and not labor-intensive technique for obtaining T cells from mouse lymph node suspensions is to overlay over a metrizamide gradient (as used for enriching in dendritic cells by Steinman and colleagues) and then continue using the pellet (which is >90% CD3+ cells) for a traditional panning over a anti-B220 coated plate. We obtained >99% CD3+ cells last week in almost 60 minutes. You can process multiple samples at the same time, which is a plus when you have many mice to start with. We used Pierce MagnaBind beads with the same antibody in parallel and we obtained very little enrichment.

What kind of sample material are you using? In case of human CD3 we provide a isolation System that can be used directly in whole blood without gradient centrifugation. We use the MEM-92 clone. The antibody MEM-92 in solution induces early responses of T cell activation (tyrosine phosphorylation, calcium elevation, Erk activation and expression of activation antigens), but it is unable to induce T cell proliferation. May it is helpful. In case you have questions or would like to have more details please contact me.

Dear Jie

thank you for your recommendation... So I am going to try it as well.  I used the Stem cell technology kit but I couldnot get rid of the other cells,..