140-004-946.01 1. Warnings 1.1 These precautions are recommended to avoid deposits in plumbing in plumbing to avoiddeposits are recommended precautions These 1. Description 4. This product is for research use only.use is forresearch product This

Contents 2. ● ● ● Components Clone Capacity 3. where explosive conditions may develop. develop. may conditions explosive where compounds should be diluted with running water before discarding. discarding. before water running with diluted be should compounds Reagents contain sodium azide. Under acidic conditions sodium sodium conditions Under acidic azide. sodium contain Reagents Product format @ macs azide yields hydrazoic acid, which is extremely toxic. Azide Azide toxic. extremely is which acid, hydrazoic yields azide Storage www.miltenyibiotec.com www.miltenyibiotec.com Friedrich-Ebert-Straße Phone Phone Miltenyi Biotec GmbH ● ● A References

1.4 1.3 Applications 1.2 1.1 Background information Background Examples of immunofluorescent staining with Anti-TOX with staining of immunofluorescent Examples staining for immunofluorescent protocol General Description DNA-binding protein family, and also known as thymocyte thymocyte as known also and family, protein DNA-binding Expression patterns: Clone REA473 recognizes the human and and human the recognizes REA473 Clone patterns: Expression mouse TOX antigen, a member of an evolutionarily conserved conserved evolutionarily of an amember TOXmouse antigen, Synonym: TOX1; KIAA0808 selection-associated HMG box protein. TOX is a thymic TOX athymic is protein. box HMG selection-associated transcript that is highly upregulated in CD4 in upregulated highly is that transcript antibodies +49 2204 8306-0, 2204 +49 miltenyibiotec.de miltenyibiotec.de ntigen: TOX ntigen:

Recommended antibody dilution dilution antibody Recommended information Background egn requirements Reagent Monoclonal Monoclonal

Store protected from light at 2−8 light from Store protected Antibodies are supplied in buffer containing containing buffer in supplied are Antibodies 300 µL: 30 tests or up 3 to tests 30 µL: 300 1 mL: 100 tests or up 10 to 100 tests 1 mL: REA473 (isotype control: REA Control (I) Control REA control: (isotype REA473 vial label. vial freeze. The expiration date is indicated on the on indicated is date expiration The freeze. conjugated to: conjugated mouse stabilizer and 0.05% sodium azide. 0.05% sodium and stabilizer antibodies). 68, 5142968, Fax Fax APC Conjugate PE +49 2204 85197 2204 +49 Bergisch Gladbach, Germany Bergisch Gladbach, Anti-TOX 130-107-785 130-107-784 1 mL (100 tests) (100 Order no. no. Order antibodies, human and and human antibodies, × total cells cells ⁸ total 10 ⁷ total cells. + CD8 130-107-838 130-107-837 (30 tests) (30 µL 300 Order no. no. Order °C. Do not Do °C. + double double 2303 2303 @ macs page 1/2 Phone Phone Miltenyi Biotec Inc. Volumes given below are for up to 10▲ Volumes are below given 1.4 cells. of formaldehyde-fixed staining for suited is antibody The Applications 1.2 The recommended antibody dilution for all Anti- all for dilution antibody recommended The 1.3 2. human and mouse and human Anti-TOX antibodies ▲ ● ● ● volumes and total volumes). total and volumes working with fewer than 10 than fewer with working reagent volumes and total volumes accordingly (e.g. for 2×10 accordingly volumes total and volumes reagent nucleated cells, use twice the volume of all indicated reagent reagent indicated of all volume the twice use cells, nucleated subsequent analysis by flow cytometry. cytometry. by flow analysis subsequent is is indicated. When working with higher cell numbers, scale up all up all scale numbers, cell higher with working When indicated.

● 1:11 for up to 10

Lindbergh Street, Auburn,Lindbergh CA Always prepare solutions freshly and according to the data sheet supplied with with supplied sheet data the to according and freshly solutions prepare Always ▲ ▲ TOX4 is known to interact with a phosphatase complex complex aphosphatase with interact to TOX4 known is Reagent requirements Reagent Recommended antibody dilution dilution antibody Recommended TOX- of enumeration and Identification Buffer: Prepare a solution containing phosphate-buffered phosphate-buffered containing solution a Prepare Buffer: General protocolGeneral for immunofluorescent (# 130 (# citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). . dextrose phosphate (ACD-A) citrate or formula-A dextrose citrate FoxP3 Staining Buffer Set (# Set 130-093-142). Buffer Staining FoxP3 (EU Hazard Classification: Xn harmful; R40/20/21/22-43). harmful; Xn Classification: Hazard (EU cell subsets. TOX expression is also detected in other tissues, tissues, other in detected also is TOX expression subsets. cell of proteins including TOX2, TOX3, and TOX4, all of which of which TOX4, TOX3, all and TOX2, including of proteins regulating metabolic processes. It is part of the larger HMG- larger of the It part is processes. metabolic regulating negligible binding to Fc to receptors. binding negligible functions of TOX have been best characterized, TOX2 may may TOX2 characterized, of TOX best have been functions specific regulated manner and in several immune-relevant immune-relevant several in and manner regulated specific saline (PBS), pH saline vivo in the While species. vertebrate in conserved highly are and 2 mM EDTA by diluting MACS® BSA Stock Solution MACS® Solution Stock BSA EDTA by diluting mM 2 and staining progression. Additional information: Clone REA473 displays displays REA473 Clone information: Additional progression. implicated been has TOX3 organs, reproductive play arole in positive thymocytes. It is expressed in the thymus in astage- in thymus the in It expressed is thymocytes. positive involved in the control of chromatin structure and cell cycle cycle cell and structure of chromatin control the in involved and cancer, breast and survival cell of neuron regulation in including liver, where it likely plays additional functions in in functions additional plays it liver, likely where including the kit. the Buffers or media containing Ca containing media or Buffers box superfamily of proteins, but also defines a small subfamily subfamily asmall defines but also of proteins, superfamily box 800 FOR MACS FOR 800 miltenyibiotec.com miltenyibiotec.com ▲ ▲

Note: Note: Cauti Note: 130 ‑ ‑

on: on: 091 091 EDTA can be replaced by other supplements such as anticoagulant anticoagulant as such supplements other by replaced be can EDTA Use o Use Items within the FoxP3 Staining Buffer Set contain formaldehyde formaldehyde contain Set Buffer Staining FoxP3 the within Items ‑ ‑ 222). Keep buffer cold 222).(2−8 °C). buffer Keep 376) 1:20 with autoMACS® Rinsing Solution Solution autoMACS®376) Rinsing with 1:20 f the FoxP3 Staining Buffer Set is critical for optimal results. results. optimal for critical is Set Buffer Staining FoxP3 f the , +1 8871, 530 888 6 cells/100 µL cells/100 7.2, 0.5% bovine serum albumin (BSA), albumin serum 7.2, bovine 0.5% 95602 2+ 6 or Mg or cells, use the same volumes as as volumes same the use cells, Fax Fax of buffer for labeling of cells and cells of labeling for of buffer , USA +1 877 591 1060 2+ are not recommended for use. for recommended not are 6 nucleated cells. When When cells. nucleated producing cells. cells. producing TOX conjugates conjugates 6 ▲ For optimal intracellular staining the FoxP3 Staining Buffer Set 2. References (# 130-093-142) must be used. Always prepare reagents freshly as 1. Yun, S. et al. (2011) TOX regulates the differentiation of human natural killer recommended in the data sheet. cells from hematopoietic stem cells in vitro. Immunol. Lett. 136 (1): 29–36. ▲ Higher temperatures and/or longer incubation times may lead 2. Aliahmad, P. et al. (2012) The many roles of TOX in the immune system. Curr. to non-specific cell labeling. Working on ice requires increased Opin. Immunol. 24 (2): 173–177. incubation times. 3. Morimura, S. et al. (2014) TOX expression in different subtypes of cutaneous lymphoma. Arch. Dermatol. Res. 306 (9): 843–849.

1. Determine cell number. Refer to www.miltenyibiotec.com for all data sheets and protocols. 2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate Warranty supernatant completely. The products sold hereunder are warranted only to be free from defects in 3. Resuspend up to 10⁶ nucleated cells in 1 mL of cold, freshly workmanship and material at the time of delivery to the customer. Miltenyi Biotec GmbH makes no warranty or representation, either expressed or implied, with prepared Fixation/Permeabilization Solution. respect to the fitness of a product for a particular purpose. There are no warranties, 4. Mix well and incubate for 30 minutes in the dark in the expressed or implied, which extend beyond the technical specifications of the products. Miltenyi Biotec GmbH’s liability is limited to either replacement of the refrigerator (2−8 °C). products or refund of the purchase price. Miltenyi Biotec GmbH is not liable for any 5. Wash cells by adding 1−2 mL of cold buffer per 10⁶ cells and property damage, personal injury or economic loss caused by the product. centrifuge at 300×g for 5 minutes at 4 °C. Aspirate supernatant autoMACS, MACS, and MACSQuant are either registered trademarks or trademarks completely. of Miltenyi Biotec GmbH. 6. Wash cells by adding 1−2 mL of cold 1× Permeabilization Copyright © 2016 Miltenyi Biotec GmbH. All rights reserved. Buffer per 10⁶ cells and centrifuge at 300×g for 5 minutes at 4 °C. Aspirate supernatant completely. 7. Resuspend up to 10⁶ nucleated cells in 100 μL of cold 1× Permeabilization Buffer. 8. Add 10 μL of the Anti-TOX antibody. 9. Mix well and incubate for 30 minutes in the dark in the refrigerator (2−8 °C). 10. Wash cells by adding 1−2 mL of cold 1× Permeabilization Buffer per 10⁶ cells and centrifuge at 300×g for 5 minutes at 4 °C. Aspirate supernatant completely. 11. Resuspend cell pellet in a suitable amount of buffer for analysis by flow cytometry or fluorescence microscopy. ▲▲Note: Due to fixation and permeabilization, cells are smaller than viable cells. Thus, FSC/SSC settings of the flow cytometer might need to be adjusted.

3. Examples of immunofluorescent staining with Anti-TOX antibodies Thymocytes from C57BL/6 mice were stained with CD4 and CD8a antibodies and fixed and permeabilized using the FoxP3 Staining Buffer Set. Cells were then stained with Anti-TOX antibodies or with the corresponding REA Control (I) antibodies (left peak) and analyzed with the MACSQuant® Analyzer. CD4+CD8low thymocytes are shown in the histogram. Cell debris were excluded from the analysis based on scatter signals.

e cell number Relativ

-1 01 10¹10² 10³ Anti-TOX-PE

For more examples please refer to the respective product page at www.miltenyibiotec.com/antibodies.

Unless otherwise specifically indicated, all Miltenyi Biotec products and services page 2/2 are for research use only and not for diagnostic or therapeutic use. 140-004-946.01