The RosetteSep™ Human Progenitor Cell Basic Pre-Enrichment Kit is designed to pre-enrich hematopoietic progenitor cells from cord blood by negative selection. Unwanted cells are targeted for removal with antibody complexes recognizing CD3, CD11b, CD19 and glycophorin A on red blood cells (RBCs). When centrifuged over a buoyant density medium such as Lymphoprep™ (a href="https://www.stemcell.com/products/lymphoprep.html">Catalog #18060), the unwanted cells pellet along with the RBCs. The hematopoietic progenitor cells are present as an enriched population at the interface between the plasma and the buoyant density medium.
Cells of interest can then be positively selected from the pre-enriched cell population using EasySep™ (e.g. EasySep™ Human CD34 Positive Selection).
Cells of interest can then be positively selected from the pre-enriched cell population using EasySep™ (e.g. EasySep™ Human CD34 Positive Selection).
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Protocols and Documentation
Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.
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This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.
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Workflow Stages for Human Pluripotent Stem Cell Research
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Frequently Asked Questions
What is RosetteSep™?
RosetteSep™ is a rapid cell separation procedure for the isolation of purified cells directly from whole blood, without columns or magnets.
How does RosetteSep™ work?
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™).
What factors affect cell recovery?
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.
Which cell samples can RosetteSep™ be used with?
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 100 RBCs for every nucleated cell (RBCs can be added if necessary).
Can RosetteSep™ be used with previously frozen or cultured cells?
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.
Can RosetteSep™ be used to enrich progenitors from cord blood?
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.
Does RosetteSep™ work with mouse cells?
No, but we have developed EasySep™, a magnetic-based cell isolation system which works with mouse and other non-human species.
Which anticoagulant should be used with RosetteSep™?
Peripheral blood should be collected in heparinized Vacutainers. Cord blood should be collected in ACD.
Should the anticoagulant be washed off before using RosetteSep™?
No, the antibody cocktail can be added directly to the sample.
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