RosetteSep® Human CD4+ T Cell Enrichment Cocktail

Procedures and instruction manuals:

•  PRODUCT INFORMATION SHEET - 15062, Lot# All:28571-PIS_1_2_0.pdf
•  PRODUCT INFORMATION SHEET - 15022, Lot# All:28571-PIS_1_2_0.pdf

Educational resources:

•  VIDEO: RosetteSep™: Cell Isolation Directly From Whole Blood Without Columns or Magnets
•  VIDEO: RosetteSep®: A Guide to Isolating NK Cells Directly from Whole Blood
•  BROCHURE: One-Step Cell Enrichment Directly From Whole Blood
•  BROCHURE: Cell Separation for Autoimmunity Research
•  WALLCHART: Antigen Processing and Presentation Wallchart
•  BROCHURE: Ready Sep Go – Cell Separation In As Little As 25 Minutes
•  SCIENTIFIC POSTER: One-step enrichment of leukocyte subsets directly in the blood collection tube
•  VIDEO: RosetteSep® for HLA Analysis
•  TECH BULLETIN: Techinical Note: Human Regulatory T Cells (Catalog #29147)

MSDS:

•  15012C.1_15022C.1-MSDS.pdf

FAQS:

• 
  HOW ROSETTESEP™ WORKS
  
  
  • Q. WHAT IS ROSETTESEP™?
    A. RosetteSep™ is a rapid cell separation procedure for the isolation of purified cells directly from whole blood, without columns or magnets.
  • Q. HOW DOES ROSETTESEP™ WORK?
    A. The antibody cocktail crosslinks unwanted cells to red blood cells (RBCs), forming rosettes. The unwanted cells then pellet with the free RBCs when centrifuged over a density centrifugation medium (e.g. Ficoll-Paque™ PLUS, Lymphoprep™).
  • Q. WHAT FACTORS AFFECT CELL RECOVERY?
    A. The temperature of the reagents can affect cell recovery. All reagents should be at room temperature (sample, density centrifugation medium, PBS, centrifuge) before performing the isolations. Layering can also affect recovery so be sure to carefully layer the sample to avoid mixing with the density centrifugation medium as much as possible. Be sure to collect the entire enriched culture without disturbing the RBC pellet. A small amount of density centrifugation medium can be collected without worry.
  
  CELL TYPES AND SAMPLE SOURCES
  
  
  • Q. WHICH CELL SAMPLES CAN ROSETTESEP™ BE USED WITH?
    A. RosetteSep™ can be used with leukapheresis samples, bone marrow or buffy coat, as long as: the concentration of cells does not exceed 5 x 10⁷ per mL (can dilute if necessary); and there are at least 20 RBCs for every nucleated cell (RBCs can be added if necessary).
  • Q. CAN ROSETTESEP™ BE USED WITH PREVIOUSLY FROZEN OR CULTURED CELLS?
    A. Yes. Cells should be re-suspended at 2 - 5 x 10⁷ cells / mL in PBS + 2% FBS. Fresh whole blood should be added at 250 µL per mL of sample, as a source of red cells.
  • Q. CAN ROSETTESEP™ BE USED TO ENRICH PROGENITORS FROM CORD BLOOD?
    A. Yes. Sometimes cord blood contains immature nucleated red cells that have a lower density than mature RBCs. These immature red cells do not pellet over Ficoll™, which can lead to a higher RBC contamination than peripheral blood separations.
  • Q. DOES ROSETTESEP™ WORK WITH MOUSE CELLS?
    A. No, but we have developed EasySep™, a magnetic-based cell isolation system which works with mouse and other non-human species.
  
  ANTI-COAGULANTS
  
  
  • Q. WHICH ANTICOAGULANT SHOULD BE USED WITH ROSETTESEP™?
    A. Peripheral blood should be collected in heparinized Vacutainers. Cord blood should be collected in ACD.
  • Q. SHOULD THE ANTICOAGULANT BE WASHED OFF BEFORE USING ROSETTESEP™?
    A. No, the antibody cocktail can be added directly to the sample.

This product has been used in:

1. Todd M Brusko et al. Human antigen-specific regulatory T cells generated by T cell receptor gene transfer.PLoS One 5 (7) e11726 (2010)
2. Anke Specht et al. Counteraction of HLA-C-mediated immune control of HIV-1 by Nef.J Virol 84 (14) 7300-7311 (July 2010)
3. Rachel A Harry et al. Generation and characterisation of therapeutic tolerogenic dendritic cells for rheumatoid arthritis.Ann Rheum Dis (June 15, 2010)
4. Laurence Weiss et al. In vivo expansion of naive and activated CD4+CD25+FOXP3+ regulatory T cell populations in interleukin-2-treated HIV patients.Proc Natl Acad Sci U S A 107 (23) 10632-10637 (June 8, 2010)
5. Jennifer M. Pfaff et al. HIV-1 resistance to CCR5 antagonists associated with highly efficient use of CCR5 and altered tropism on primary CD4+ T cells.J. Virol. 84 (13) JVI.00374-10-6514 (April 21, 2010)
6. Micha??l Imbeault et al. Microarray study reveals that HIV-1 induces rapid type-I interferon-dependent p53 mRNA up-regulation in human primary CD4+ T cells.Retrovirology 6 5 (2009)
7. Nicholas J Megjugorac et al. Modulation of human plasmacytoid DC function by IFN-lambda1 (IL-29).J Leukoc Biol 86 (6) 1359-1363 (December 2009)
8. Khoa D Nguyen et al. Impaired IL-10-dependent induction of tolerogenic dendritic cells by CD4+CD25hiCD127lo/- natural regulatory T cells in human allergic asthma.Am J Respir Crit Care Med 180 (9) 823-833 (November 1, 2009)
9. Jeffrey C Nolz et al. TCR/CD28-stimulated actin dynamics are required for NFAT1-mediated transcription of c-rel leading to CD28 response element activation.J Immunol 179 (2) 1104-1112 (July 15, 2007)
10. Stella A Nicolaou et al. Altered dynamics of Kv1.3 channel compartmentalization in the immunological synapse in systemic lupus erythematosus.J Immunol 179 (1) 346-356 (July 1, 2007)
11. Jill F. Wright et al. Identification of an interleukin 17F/17A heterodimer in activated human CD4+ T cells.J Biol Chem 282 (18) 13447-13455 (May 4, 2007)
12. Nicole L. Yonkers et al. TLR Ligand-Dependent Activation of Naive CD4 T Cells by Plasmacytoid Dendritic Cells Is Impaired in Hepatitis C Virus InfectionJ Immunol 178 (7) 4436-4444 (April 1, 2007)
13. Haviva Veler et al. Superantigen presentation by airway smooth muscle to CD4+ T lymphocytes elicits reciprocal proasthmatic changes in airway function.J Immunol 178 (6) 3627-3636 (March 15, 2007)
14. Jeroen P Roose et al. Unusual Interplay of Two Types of Ras Activators, RasGRP and SOS, Establishes Sensitive and Robust Ras Activation in Lymphocytes.Mol Cell Biol 27 (7) 2732-2745 (February 5, 2007)
15. M Leiba et al. Halofuginone inhibits NF-kappaB and p38 MAPK in activated T cells.J Leukoc Biol 80 (2) 399-406 (August 2006)
16. Steffen Wildum et al. Contribution of Vpu, Env, and Nef to CD4 down-modulation and resistance of human immunodeficiency virus type 1-infected T cells to superinfection.J Virol 80 (16) 8047-8059 (August 2006)
17. Alexandra Zanin-Zhorov et al. Heat shock protein 60 enhances CD4+ CD25+ regulatory T cell function via innate TLR2 signaling.J Clin Invest 116 (7) 2022-2032 (July 2006)
18. Julia Yuen-Shan Tsang et al. Altered proximal T cell receptor (TCR) signaling in human CD4+CD25+ regulatory T cells.J Leukoc Biol 80 (1) 145-151 (July 2006)
19. Weihong Liu et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4(+) T reg cells.J Exp Med 203 (7) 1701-1711 (July 10, 2006)
20. Jennifer A Fulcher et al. Galectin-1-matured human monocyte-derived dendritic cells have enhanced migration through extracellular matrix.J Immunol 177 (1) 216-226 (July 1, 2006)
21. Louise Swainson et al. IL-7R alpha gene expression is inversely correlated with cell cycle progression in IL-7-stimulated T lymphocytes.J Immunol 176 (11) 6702-6708 (June 1, 2006)
22. Luciana Lorena Molinero et al. Intracellular expression of MICA in activated CD4 T lymphocytes and protection from NK cell-mediated MICA-dependent cytotoxicity.Hum Immunol 67 (3) 170-182 (March 2006)
23. Kayoko Sato et al. TRAIL-expressing T cells induce apoptosis of vascular smooth muscle cells in the atherosclerotic plaque.J Exp Med 203 (1) 239-250 (January 23, 2006)
24. Jens M Chemnitz et al. Prostaglandin E2 impairs CD4+ T cell activation by inhibition of lck: implications in Hodgkin's lymphoma.Cancer Res 66 (2) 1114-1122 (January 15, 2006)
25. Randy Q Cron et al. Early growth response-1 is required for CD154 transcription.J Immunol 176 (2) 811-818 (January 15, 2006)
26. Hui Fang et al. Anthrax lethal toxin blocks MAPK kinase-dependent IL-2 production in CD4+ T cells.J Immunol 174 (8) 4966-4971 (April 15, 2005)
27. John F Foley et al. Roles for CXC chemokine ligands 10 and 11 in recruiting CD4+ T cells to HIV-1-infected monocyte-derived macrophages, dendritic cells, and lymph nodes.J Immunol 174 (8) 4892-4900 (April 15, 2005)
28. Hidenori Sasaki et al. Overexpression of a cell adhesion molecule, TSLC1, as a possible molecular marker for acute-type adult T-cell leukemia.Blood 105 (3) 1204-1213 (February 1, 2005)
29. Juli?? Blanco et al. High level of coreceptor-independent HIV transfer induced by contacts between primary CD4 T cells.J Biol Chem 279 (49) 51305-51314 (December 3, 2004)
30. Laurence Weiss et al. Human immunodeficiency virus-driven expansion of CD4+CD25+ regulatory T cells, which suppress HIV-specific CD4 T-cell responses in HIV-infected patients.Blood 104 (10) 3249-3256 (November 15, 2004)
31. Alexandra Trkola et al. Human immunodeficiency virus type 1 fitness is a determining factor in viral rebound and set point in chronic infection.J Virol 77 (24) 13146-13155 (December 2003)
32. Ronald L Rabin et al. CXCR3 is induced early on the pathway of CD4+ T cell differentiation and bridges central and peripheral functions.J Immunol 171 (6) 2812-2824 (September 15, 2003)
33. Jason A LaBonte et al. Cytolysis by CCR5-using human immunodeficiency virus type 1 envelope glycoproteins is dependent on membrane fusion and can be inhibited by high levels of CD4 expression.J Virol 77 (12) 6645-6659 (June 2003)
34. Szun S Tay et al. IFN-gamma reverses the stop signal allowing migration of antigen-specific T cells into inflammatory sites.J Immunol 170 (6) 3315-3322 (March 15, 2003)
35. Els Verhoeyen et al. IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes.Blood 101 (6) 2167-2174 (March 15, 2003)
36. M M Addo et al. Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load.J Virol 77 (3) 2081-2092 (February 2003)
37. Claire E Hirst et al. The intracellular granzyme B inhibitor, proteinase inhibitor 9, is up-regulated during accessory cell maturation and effector cell degranulation, and its overexpression enhances CTL potency.J Immunol 170 (2) 805-815 (January 15, 2003)
38. Udaya K Liyanage et al. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma.J Immunol 169 (5) 2756-2761 (September 1, 2002)

FACS Histogram Results Using RosetteSep® Human CD4+ T Cell Enrichment Cocktail

RosetteSep® Procedure