EasySep® Human CD4+CD127lowCD49d- Regulatory T Cell Enrichment Kit

Recommended for:	The isolation of human CD4+CD127lowCD49d- T cells from fresh peripheral blood mononuclear cells by negative selection.
Components:	• EasySep™ Human CD4+CD127lowCD49d- Regulatory T Cell Enrichment Cocktail, 1.0 mL • EasySep™ Dextran Selection Cocktail, 1.0 mL • EasySep™ Magnetic Particles, 3 x 1.0 mL
Intended Use Statement:	For Research Use Only. Not for Therapeutic or Diagnostic Use.
Contains:	Tetrameric antibody complexes against red blood cells, non-CD4+ T-cells and cells expressing high levels of CD127 and CD49d.
Equipment Required:	• EasySep™ Magnet (Catalog #18000), or • “The Big Easy” EasySep™ Magnet (Catalog #18001), or • RoboSep™ (Catalog #20000)
Advantages:	Only single step negative selection method for the isolation of untouched human regulatory T cells
Product Type:	Cell isolation products
Area of Interest:	HIV, Immunology
Cell Isolation Product Type:	Reagents
Cell Source:	PBMC
Cell Type:	CD4+ T cells, Regulatory T cells, T cells
Popular Product Line:	EasySep
Selection Method:	Negative
Species:	Human

Procedures and instruction manuals:

•  PRODUCT INFORMATION SHEET - 19232, Lot# All:28781-PIS_1_0_0.pdf
•  PRODUCT INFORMATION SHEET - 19232RF, Lot# All:28781-PIS_1_0_0.pdf

Educational resources:

•  SCIENTIFIC POSTER: Column-free isolation of untouched human regulatory T cells in 45 minutes
•  TECH BULLETIN: Techinical Note: Human Regulatory T Cells (Catalog #29147)

MSDS:

•  19202C_19232C-MSDS.pdf
•  19253C-MSDS.pdf

FAQS:

• 
  HOW EASYSEP™ WORKS
  
  
  • Q. CAN EASYSEP™ BE USED FOR EITHER POSITIVE OR NEGATIVE SELECTION?
    A. Yes. The EasySep™ kits use either a negative selection approach by targeting and removing unwanted cells or a positive selection approach targeting desired cells. Depletion kits are also available for the removal of cells with a specific undesired marker (e.g. GlyA).
  • Q. HOW DOES THE SEPARATION WORK?
    A. Magnetic particles are crosslinked to cells using Tetrameric Antibody Complexes (TAC). When placed in the EasySep™ Magnet, labeled cells migrate to the wall of the tube. The unlabeled cells are then poured off into a separate fraction.
  • Q. WHICH COLUMNS DO I USE?
    A. The EasySep™ procedure is column-free. That's right - no columns!
  • Q. HOW CAN I ANALYZE THE PURITY OF MY ENRICHED SAMPLE?
    A. The Product Information Sheet provided with each EasySep™ kit contains detailed staining information.
  • Q. CAN EASYSEP™ SEPARATIONS BE AUTOMATED?
    A. Yes. RoboSep™, the fully automated cell separator, automates all EasySep™ labeling and cell separation steps.
  • Q. CAN EASYSEP™ BE USED TO ISOLATE RARE CELLS?
    A. Yes. We recommend a cell concentration of 2x10⁸ cells/mL and a minimum working volume of 100 µL. Samples containing 2x10⁷ cells or fewer should be suspended in 100 µL of buffer.
  
  EASYSEP™ MAGNETIC PARTICLES
  
  
  • Q. ARE THE EASYSEP™ MAGNETIC PARTICLES FACS-COMPATIBLE?
    A. Yes, the EasySep™ particles are flow cytometry-compatible, as they are very uniform in size and about 5000X smaller than other commercially available magnetic beads used with column-free systems.
  • Q. CAN THE EASYSEP™ MAGNETIC PARTICLES BE REMOVED AFTER ENRICHMENT?
    A. No, but due to the small size of these particles, they will not interfere with downstream applications.
  • Q. HOW DOES THE BINDING OF THE EASYSEP™ MAGNETIC PARTICLE AFFECT THE CELLS? IS THE FUNCTION OF POSITIVELY SELECTED CELLS ALTERED BY THE BOUND PARTICLES?
    A. Hundreds of publications have used cells selected with EasySep™ positive selection kits for functional studies. Our in-house experiments also confirm that selected cells are not functionally altered by the EasySep™ magnetic particles.
    
    If particle binding is a key concern, we offer two options for negative selection. The EasySep™ negative selection kits can isolate untouched cells with comparable purities, while RosetteSep™ can isolate untouched cells directly from whole blood without using particles or magnets.
  
  MODIFYING THE EASYSEP™ PROTOCOL
  
  
  • Q. CAN I ALTER THE SEPARATION TIME IN THE MAGNET?
    A. Yes; however, this may impact the kit's performance. The provided EasySep™ protocols have already been optimized to balance purity, recovery and time spent on the isolation.
  • Q. FOR POSITIVE SELECTION, CAN I PERFORM MORE THAN 3 SEPARATIONS TO INCREASE PURITY?
    A. Yes, the purity of targeted cells will increase with additional rounds of separations; however, cell recovery will decrease.

Product Name	Description	Catalog #

Start: 1.84% 1.84% CD3+CD4+CD127lowCD49d-CD25+FOXP3+ Cells

Starting with fresh PBMCs, the CD3+CD4+CD127lowCD49d-CD25+FOXP3+ cell content of the enriched fraction typically ranges from 57.4 – 87.4%. Donor to donor variability is unavoidable and may affect the purity of the enriched fraction.

Enriched: 84.1% CD3+CD4+CD127lowCD49d-CD25+FOXP3+ Cells

Starting with fresh PBMCs, the CD3+CD4+CD127lowCD49d-CD25+FOXP3+ cell content of the enriched fraction typically ranges from 57.4 – 87.4%. Donor to donor variability is unavoidable and may affect the purity of the enriched fraction.