The best way to obtain untouched basophils is by negative bead isolation from total leukocytes (obtained with HetaSep). By using Ficoll to obtain PBMCs, you will lose part (>50%) of the basophils.

I have tested kits from Miltenyi and from Stemcell, and obtained best purities and yield with the kit from Stemcell: EasySep Human basophil enrichment kit. 

See examples from isolation in attached publication.

Article Activation of human basophils by combined Toll-like receptor...

Depends on what kind of purity you need.  The easiest way is to use a basophil isolation kit from Miltenyi Biotec or Stemcell as mentioned above by Jolien.  If you need better purity, you can try enriching first with a two-step Percoll gradient (1.065 g/mL/1.079 g/mL) and then use negative isolation with the Miltenyi or Stemcell kit.  You can probably get sample kits from each company and see which one works best in your hands. 

Here's a paper that describes the two-step percoll gradient:

In Vitro Regulation of FceRI a Expression on Human Basophils by IgE Antibody

Donald MacGlashan, et al

Blood, Vol 91, No 5 (March 1), 1998: pp 1633-1643

http://www.bloodjournal.org/content/bloodjournal/91/5/1633.full.pdf?ck=nck&sso-checked=true

I agree with Jolien-

however, we do not loose so many basophils on a Percoll gradient (Percoll is a little less toxic as compared to Ficoll) and we have less contamination with neutrophils and erythrocytes after the gradient as compared to hetasep. We use Hetasep for functional assays (when we want our cells as native as possible) without further isolation of basophils (for flow cytometry). Furthermore, we did not see phenotypical differences between basophils in the interphase and in the erythrocyte fraction!

The Stemcell-Kit gives us high purities (100 %), however, we loose a lot of cells.

The Miltenyi-Kit gives us lower purities in buffy coat blood, which is a little older (but stil 80-90%). It gives us high purities and yields in fresh blood (also 100%).

good luck,

Dorrit

HI

it all depends on the intended final purity. As stated by David Larson above, there are two immunomagnetic kits for human basophil purification, one from StemCell Technologies and one from Miltenyi Biotech. You'll have to invest in a magnet first, if you don't have one.

If you want to read a historical perspective on basophil purification, with an easy to follow step-by-step protocol using the StemCell kit, have a look at :

Falcone FH, Gibbs BF. Methods Mol Biol. Purification of basophils from peripheral human blood. 2014;1192:35-47. doi: 10.1007/978-1-4939-1173-8_2.

Also

Gibbs BF, Papenfuss K, Falcone FH. A rapid two-step procedure for the purification of human peripheral blood basophils to near homogeneity. Clin Exp Allergy. 2008 Mar;38(3):480-5. doi: 10.1111/j.1365-2222.2007.02919.x.

I would not recommend using flow cytometry for sorting basophils. Yields are very low and there can be issues about their functionality.

Hope this helps, feel free to get in touch if there are issues

Hi.

Dorritt and Franco are the best indicated Colleagues in providing you with the most excellent reply to your request. I never separated physically basophils, i prefer to "electronically" (virtally) separate cells with flow cytometry. Less expensive, less time-consuming, less boring....I know it: isolated basophils are not influenced by other cells and soluble factors but you may have gross problems on yields, spontaneous activation, cellular function as response to external stimuli but, particularly, you should investigate on cells OUT of their usual and physiological environment: whole blood. A good reply depends on what are you wishing to do with these "puzzling" leukocytes... 

Dear Colleague, I fully agree with Prof. Falcone's reply. However, physically purified basophils are very difficult to handle or manage and particularly sensitive to any membrane perturbing factor. Anyway, the methods suggested are highly better performing approaches than Cell Sorter. Flow cytometry to capture basophils (non physically) is a good, cheepar alternative to study these cells...Best wishes, Salvatore

We do not sort basophils by flow-assisted sorting for the isolation of basophils!  We do our functional assays (BAT, phosphorylation of certain targets) in PBMC (obtained by HetaSEP or after Percoll separation) and measure the respective readout by FACS.

Other functional assays are done using isolated cells- as Salvatore said: it depends on your aim.

Jolien is actually also a very good basophil researcher! ;-)

As largely indicated previously, Hetasep gradient followed by Stem cell enrichent kit are the best that I recommend to use if you are not familiar with percoll separation. 

Thank you Dorrit for your precious note about Jolien. I'm following her!  Cheers, Salvatore

I am thankful for your valuable suggestions! Because of a lack of necessary techniques as mentioned above, we just intended to achieve the highest purity as much as possible after 2 step percoll gradient (or sedimentation). Some papers yielded high basophil purity (8 to 45%) but I couldn't achieve that value. I will consider all of your suggestions. Once again many thanks to you all! I will update my results later. 

It has been shown that CD16 is on 25% of human basophils. Be careful with the magnetic bead isolation kits, as one of the antibodies included in kits is CD16. Therefore, you will remove what could be an important subset of basophils.