We currently use a Ficoll overlay procedure to isolate PBMCs from whole blood. Often we have up to twenty 10ml tubes of blood to isolate cells from multiple times a day and I am interested in streamlining our workflow by using SepMate or Leucosep tubes.
The Leucosep tubes seem more efficient since you can pour the diluted blood directly into the prepared tube, and the SepMate still needs layering since the Ficoll is above the frit and will mix when layering the blood.
Does anyone have input on either method, or done comparison between the two?
I have not used SepMate, but would definately recommend the Leucosep. You will definately save time as slow layering is not needed. They also come in different sizes which work well if you have smaller sample volumes.
Same as Sheila, I have not used SepMate, but Leucosep is our gold-standard in PBMC isolation with Histopaque 1077. Good luck
kudos to histopaque 1077... if we are using the same in 4degre rather than at room temperature, granulocyte contamination can be reduced, if incase you need pure Lymphocyte..good luck with ur exp..
Hi all,
I'm the product manager for SepMate at STEMCELL, and I wanted to reply here to provide some more information about these new tubes.
First, in response to the original question, the SepMate tube does not actually require careful layering of the blood sample: you can pour or quickly pipet the sample into the tube instead. Although the blood sample will mix with the Ficoll above the insert, this does not affect performance. One advantage SepMate has over Leucosep is that you can add the density medium directly through the central hole in the insert. (Unless you buy the pre-filled tubes, Leucosep requires an additional spin step to get the density medium below the frit.)
An additional advantage of SepMate is that it can be combined with RosetteSep, a reagent for isolating cell subsets directly from blood during density gradient centrifugation. If you work with specific cell types, this allows you to skip downstream purification steps and go from whole blood straight to purified cells in around 25 minutes.
The purpose of the SepMate tubes is to facilitate rapid and consistent PBMC isolation, and they were designed for researchers who, like you, need to process large numbers of blood samples every day. To help you with your decision, I'd be glad to arrange to send some samples over, or put you in touch with references for the product. Just send me a message (or, if you prefer, visit this URL: http://www.stemcell.com/en/Forms/Samples-and-Demos/Sample-Request-SepMate.aspx).
You may also be interested in this recent publication discussing SepMate and RosetteSep for high-throughput NK cell isolation: http://www.ncbi.nlm.nih.gov/pubmed/23680234
Best,
Mike
Chamilly Evaldsson, I had a quick question for you. Have you conducted the validation experiments you mentioned above? Keen to know your results. We are in the process of starting blood collection for an epidemiological study and being able to use a PBMC separation method that is quick would be a major bonus. However it is time over quality and therefore I am keen to know your results..
regards,
Prachee
I have used Histopaque for density gradient centrifugation to isolate PBMC from whole blood. It was for a genetic epidemiology study and was quite happy with the method. You just need to be careful with the layering of blood over the histopaque and aspiration after centrifugation. I used to get good quality yield of DNA and RNA from the PBMC thus isolated. I am not sure if it works for iPSC studies.
Hello guys, I have followed this discussion carefully. I am looking for a technique that can help us separate white blood cells from whole blood so w can do further tests for oxidation stress, apoptosis and viability. Any suggestions please.
I use SepMate, but not the Leucosep. They work much better than just layering alone. In my case, occasionally the media does not pour down to the bottom on its own, and I need to centrifuge it down. Other than that it makes things much easier
We use SepMate in our laboratory because after PBMC isolation we also need the Granulocytes which are in the bottom under the membrane (with a hole in the middle). We easily can extract the granulozytes we have same amount of PBMCs and quality as when we use a regular Ficoll/Pancoll Protokoll. I'm interested how you can get out the Granulocyte-Fraktion when using Leucosep?
Katrin Kaehlcke How do you get to the granulocytes? Im struggling when using the SepMate tubes
