Anti-Helios Purified

*Serre K, Bénézech C, Desanti G, Bobat S, Toellner KM, Bird R, Chan S, Kastner P, Cunningham AF, Maclennan IC, Mohr E: Helios is associated with CD4 T cells differentiating to T helper 2 and follicular helper T cells in vivo independently of Foxp3 expression. PLoS One. 2011;6 (6) :e20731., URL: https://pubmed.ncbi.nlm.nih.gov/21677778/, *Thornton AM, Korty PE, Tran DQ, Wohlfert EA, Murray PE, Belkaid Y, Shevach EM: Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J Immunol. 2010 Apr 1;184 (7) :3433-41., URL: https://pubmed.ncbi.nlm.nih.gov/20181882/, *McIver Z, Melenhorst JJ, Wu C, Grim A, Ito S, Cho I, Hensel N, Battiwalla M, Barrett AJ: Donor lymphocyte count and thymic activity predict lymphocyte recovery and outcomes after matched-sibling hematopoietic stem cell transplant. Haematologica. 2013 Mar;98 (3) :346-52., URL: http://www.ncbi.nlm.nih.gov/pubmed/23065508, *Atarashi K, Tanoue T, Oshima K, Suda W, Nagano Y, Nishikawa H, Fukuda S, Saito T, Narushima S, Hase K, Kim S, Fritz JV, Wilmes P, Ueha S, Matsushima K, Ohno H, Olle B, Sakaguchi S, Taniguchi T, Morita H, Hattori M, Honda K: Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature. 2013 Aug 8;500 (7461) :232-6., URL: http://www.ncbi.nlm.nih.gov/pubmed/23842501 , *Blankenhaus B, Reitz M, Brenz Y, Eschbach ML, Hartmann W, Haben I, Sparwasser T, Huehn J, Kühl A, Feyerabend TB, Rodewald HR, Breloer M. Foxp3⁺ regulatory T cells delay expulsion of intestinal nematodes by suppression of IL-9-driven mast cell activation in BALB/c but not in C57BL/6 mice. PLoS Pathog. 2014 Feb 6;10 (2) :e1003913., URL: https://pubmed.ncbi.nlm.nih.gov/24516385/, *Daley SR1, Hu DY, Goodnow CC: Helios marks strongly autoreactive CD4+ T cells in two major waves of thymic deletion distinguished by induction of PD-1 or NF-κ B. J Exp Med. 2013 Feb 11;210 (2) :269-85., URL: http://www.ncbi.nlm.nih.gov/pubmed/23337809 , *Pinheiro D, Singh Y, Grant CR, Appleton RC, Sacchini F, Walker KR, Chadbourne AH, Palmer CA, Armitage-Chan E, Thompson I, Williamson L, Cunningham F, Garden OA. Phenotypic and functional characterization of a CD4 (+) CD25 (high) FOXP3 (high) regulatory T-cell population in the dog. Immunology. 2011 Jan;132 (1) :111-22., URL: http://www.ncbi.nlm.nih.gov/pubmed/20880379 , *Fourcade J, Sun Z, Chauvin JM, Ka M, Davar D, Pagliano O, Wang H, Saada S, Menna C, Amin R, Sander C, Kirkwood JM, Korman AJ, Zarour HM: CD226 opposes TIGIT to disrupt Tregs in melanoma. JCI Insight. 2018 Jul 26;3 (14) :e121157. , URL: https://pubmed.ncbi.nlm.nih.gov/30046006/, *Černý V, Novotná O, Petrásková P Hudcová K, Boráková K, Prokešová L, Kolářová L, Hrdý J: Lower functional and proportional characteristics of cord blood treg of male newborns compared with female newborns. Biomedicines 2021, 9, 170., URL: https://pubmed.ncbi.nlm.nih.gov/33572097/

The Armenian hamster monoclonal antibody 22F6 recognizes Helios, a transcription factor (intracellular antigen) expressed in some hematopoietic stem cells, and at high levels in thymic-derived regulatory T cells. The epitope is located between amino acids 51 and 107.

Flow cytometry: Recommended dilution: 1-4 μg/ml., Recommended protocol: 1) Perform staining of cell surface markers (CD25, CD4 etc.) for 20 min. at room temperature in the dark; 100 μl of peripheral blood. 2) Add 3 ml of PBS with 1% BSA, centrifugate at 300g and discard the supernatant. Further steps perform on ice and with ice-cold reagents. 3) Resuspend the cells in 5 ml of cold fixation solution (Miltenyi Biotec) and incubate for 30 min. on ice. 4) Centrifugate for 5 min. at 1000 g, 4°C, and discard the supernatant. 5) Resuspend the cells in 5 ml of ice-cold PBS with 1% BSA. 6) Centrifugate for 5 min. at 1000 g, 4°C, and discard the supernatant. 7) Resuspend the cells in 5 ml of ice-cold permeabilization solution (Miltenyi Biotec) and incubate 5 min. 8) Centrifugate for 5 min. at 1000 g, 4°C, and discard the supernatant. 9) Resuspend the cells in μl of ice-cold permeabilization solution and add 20 μl of FcR blocking solution and incubate for 5 min. (4°C, in the dark) . 10) Perform intracellular staining of Helios for 30 min. (4°C, in the dark) with 10 μl of anti-Helios PE antibody (22F6) in 100 μl. 11) Add 2 mLof ice-cold permeabilization solution and incubate for 5 min. at 4°C in the dark. 12) Centrifugate for 5 min. at 1000 g, 4°C, and discard the supernatant. 13) Resuspend the cells in 3 ml of ice-cold PBS with 1% BSA. 14) Centrifugate for 5 min. at 1000 g, 4°C, and discard the supernatant. 15) Resuspend the cells in 150 μl of ice-cold PBS with 1% BSA and measure on a flow cytometry device with appropriate setting as soon as possible. Keep cold until measuring.