RosetteSep™ Human CD8+ T Cell Enrichment Cocktail

Procedures and instruction manuals:

•  PRODUCT INFORMATION SHEET - 15063, Lot# 11F39695 or greater:28572-PIS_1_3_0.pdf
•  PRODUCT INFORMATION SHEET - 15063, Lot# 11F39694 or less:28572-PIS_1_2_0.pdf
•  PRODUCT INFORMATION SHEET - 15023, Lot# 11F39696 or greater:28572-PIS_1_3_0.pdf
•  PRODUCT INFORMATION SHEET - 15023, Lot# 11F39695 or less:28572-PIS_1_2_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
•  SCIENTIFIC POSTER: One-step enrichment of leukocyte subsets directly in the blood collection tube
•  VIDEO: RosetteSep® for HLA Analysis

MSDS:

•  15013C.2_15023C.2-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. Anita Parmigiani et al. Interleukin-21 and cellular activation concurrently induce potent cytotoxic function and promote antiviral activity in human CD8 T cells.Hum Immunol (October 25, 2010)
2. Melany J Wagner et al. Herpes simplex virus requires VP11/12 to induce phosphorylation of the activation loop tyrosine (Y394) of the Src family kinase Lck in T lymphocytes.J Virol 83 (23) 12452-12461 (December 2009)
3. 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)
4. Xiao-Nong Wang et al. Regulatory T-cell suppression of CD8+ T-cell-mediated graft-versus-host reaction requires their presence during priming.Transplantation 88 (2) 188-197 (July 27, 2009)
5. 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)
6. M Leiba et al. Halofuginone inhibits NF-kappaB and p38 MAPK in activated T cells.J Leukoc Biol 80 (2) 399-406 (August 2006)
7. 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)
8. Mathias Lichterfeld et al. Loss of HIV-1???specific CD8+ T Cell Proliferation after Acute HIV-1 Infection and Restoration by Vaccine-induced HIV-1???specific CD4+ T CellsThe Journal of Experimental Medicine 200 (6) (September 20, 2004)
9. Tonie Cilliers et al. The CCR5 and CXCR4 Coreceptors Are Both Used by Human Immunodeficiency Virus Type 1 Primary Isolates from Subtype CJ. Virol. 77 (7) 4449-4456 (April 1, 2003)
10. 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)
11. 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)
12. 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 CD8+ T Cell Enrichment Cocktail

RosetteSep® Procedure