RosetteSep™ Human NK Cell Enrichment Cocktail

Procedures and instruction manuals:

•  PRODUCT INFORMATION SHEET - 15065, Lot# 11F39744 or greater:28574-PIS_1_1_0.pdf
•  PRODUCT INFORMATION SHEET - 15065, Lot# 11F39743 or less:28574-PIS_1_0_0.pdf
•  PRODUCT INFORMATION SHEET - 15025, Lot# 11F39745 or greater:28574-PIS_1_1_0.pdf
•  PRODUCT INFORMATION SHEET - 15025, Lot# 11F39744 or less:28574-PIS_1_0_0.pdf

Educational resources:

•  VIDEO: RosetteSep® Introduction
•  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
•  VIDEO: RosetteSep® for HLA Analysis
•  BROCHURE: Fast and Easy Isolation of Natural Killer Cells

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. Eun-Ok Kim et al. Homotypic cell to cell cross-talk among human natural killer cells reveals differential and overlapping roles of 2B4 and CD2.J Biol Chem 285 (53) 41755-41764 (December 31, 2010)
2. Ashley N Mentlik et al. Rapid lytic granule convergence to the MTOC in natural killer cells is dependent on dynein but not cytolytic commitment.Mol Biol Cell 21 (13) 2241-2256 (July 2010)
3. Michela Falco et al. Combined genotypic and phenotypic killer cell Ig-like receptor analyses reveal KIR2DL3 alleles displaying unexpected monoclonal antibody reactivity: identification of the amino acid residues critical for staining.J Immunol 185 (1) 433-441 (July 1, 2010)
4. Mariella Della Chiesa et al. GPR56 as a novel marker identifying the CD56dull CD16+ NK cell subset both in blood stream and in inflamed peripheral tissues.Int Immunol 22 (2) 91-100 (February 2010)
5. Jianhua Yu et al. CD94 surface density identifies a functional intermediary between the CD56bright and CD56dim human NK-cell subsets.Blood 115 (2) 274-281 (November 6, 2009)
6. Carolina I Domaica et al. Tumour-experienced T cells promote NK cell activity through trogocytosis of NKG2D and NKp46 ligands.EMBO Rep 10 (8) 908-915 (August 2009)
7. Sheng Wei et al. A critical role for phosphatase haplodeficiency in the selective suppression of deletion 5q MDS by lenalidomide.Proc Natl Acad Sci U S A 106 (31) 12974-12979 (August 4, 2009)
8. Daniela Pende et al. Anti-leukemia activity of alloreactive NK cells in KIR ligand-mismatched haploidentical HSCT for pediatric patients: evaluation of the functional role of activating KIR and re-definition of inhibitory KIR specificity.Blood 113 (13) 3119-3129 (October 22, 2008)
9. Rossana Trotta et al. TGF-beta utilizes SMAD3 to inhibit CD16-mediated IFN-gamma production and antibody-dependent cellular cytotoxicity in human NK cells.J Immunol 181 (6) 3784-3792 (September 15, 2008)
10. Mercedes B Fuertes et al. Intracellular retention of the NKG2D ligand MHC class I chain-related gene A in human melanomas confers immune privilege and prevents NK cell-mediated cytotoxicity.J Immunol 180 (7) 4606-4614 (April 1, 2008)
11. Mar??a V Girart et al. Engagement of TLR3, TLR7, and NKG2D regulate IFN-gamma secretion but not NKG2D-mediated cytotoxicity by human NK cells stimulated with suboptimal doses of IL-12.J Immunol 179 (6) 3472-3479 (September 15, 2007)
12. Yong Tang et al. Regulation of antibody-dependent cellular cytotoxicity by IgG intrinsic and apparent affinity for target antigen.J Immunol 179 (5) 2815-2823 (September 1, 2007)
13. Loredana Ruggeri et al. Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value.Blood 110 (1) 433-440 (July 1, 2007)
14. Roberta Castriconi et al. Functional characterization of natural killer cells in type I leukocyte adhesion deficiency.Blood 109 (11) 4873-4881 (June 1, 2007)
15. Derin B Keskin et al. TGFbeta promotes conversion of CD16+ peripheral blood NK cells into CD16- NK cells with similarities to decidual NK cells.Proc Natl Acad Sci U S A 104 (9) 3378-3383 (February 27, 2007)
16. Paola Vacca et al. Analysis of natural killer cells isolated from human decidua: Evidence that 2B4 (CD244) functions as an inhibitory receptor and blocks NK-cell function.Blood 108 (13) 4078-4085 (December 15, 2006)
17. Stefania Marcenaro et al. Analysis of natural killer-cell function in familial hemophagocytic lymphohistiocytosis (FHL): defective CD107a surface expression heralds Munc13-4 defect and discriminates between genetic subtypes of the disease.Blood 108 (7) 2316-2323 (October 1, 2006)
18. Mariella Della Chiesa et al. The tryptophan catabolite L-kynurenine inhibits the surface expression of NKp46- and NKG2D-activating receptors and regulates NK-cell function.Blood 108 (13) 4118-4125 (August 10, 2006)
19. Xi Chen et al. CD28-stimulated ERK2 phosphorylation is required for polarization of the microtubule organizing center and granules in YTS NK cells.Proc Natl Acad Sci U S A 103 (27) 10346-10351 (July 5, 2006)
20. Julie M Roda et al. Interleukin-21 enhances NK cell activation in response to antibody-coated targets.J Immunol 177 (1) 120-129 (July 1, 2006)
21. Stefania Fontana et al. Innate immunity defects in Hermansky-Pudlak type 2 syndrome.Blood 107 (12) 4857-4864 (June 15, 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. Chiho Goda et al. Involvement of IL-32 in activation-induced cell death in T cells.Int Immunol 18 (2) 233-240 (February 2006)
24. Christelle Cebo et al. The decreased susceptibility of Bcr/Abl targets to NK cell-mediated lysis in response to imatinib mesylate involves modulation of NKG2D ligands, GM1 expression, and synapse formation.J Immunol 176 (2) 864-872 (January 15, 2006)
25. Aleksandra Bielawska-Pohl et al. Human NK cells lyse organ-specific endothelial cells: analysis of adhesion and cytotoxic mechanisms.J Immunol 174 (9) 5573-5582 (May 1, 2005)
26. Ala Aoukaty et al. Role for glycogen synthase kinase-3 in NK cell cytotoxicity and X-linked lymphoproliferative disease.J Immunol 174 (8) 4551-4558 (April 15, 2005)
27. Rossana Trotta et al. Differential expression of SHIP1 in CD56bright and CD56dim NK cells provides a molecular basis for distinct functional responses to monokine costimulation.Blood 105 (8) 3011-3018 (April 15, 2005)
28. Alessandro Poggi et al. Tumor-induced apoptosis of human IL-2-activated NK cells: role of natural cytotoxicity receptors.J Immunol 174 (5) 2653-2660 (March 1, 2005)
29. Xiao-Song He et al. T cell-dependent production of IFN-gamma by NK cells in response to influenza A virus.J Clin Invest 114 (12) 1812-1819 (December 2004)
30. Shuji Miyagawa et al. Delta-short consensus repeat 4-decay accelerating factor (DAF: CD55) inhibits complement-mediated cytolysis but not NK cell-mediated cytolysis.J Immunol 173 (6) 3945-3952 (September 15, 2004)
31. Jodie P Goodridge et al. KIR2DL4 (CD158d) genotype influences expression and function in NK cells.J Immunol 171 (4) 1768-1774 (August 15, 2003)
32. Huei-Wei Chan et al. DNA methylation maintains allele-specific KIR gene expression in human natural killer cells.J Exp Med 197 (2) 245-255 (January 20, 2003)
33. 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)

FACS Histogram Results Using RosetteSep® Human NK Cell Enrichment Cocktail

RosetteSep® Procedure