GMP Cloudz Human Treg Cell Expansion Kit
GMP Cloudz Human Treg Cell Expansion Kit Summary
Key Benefits
• > 250-fold expansion of human regulatory T (Treg) Cells from CD4+ T cells in 9 days
• Dissolvable CD3/28 microspheres enable Treg cell expansions
• Optimized for use with the ExCellerate™ Human T Cell Media
• Supports Treg cell expansion from CD4-positive cells
• Optimized for use with R&D Systems cytokines and media
Why Expand Treg Cells ex vivo Using GMP Cloudz Human Treg Cell Expansion Kit?
Treg cells are a suppressive subset of CD4+ T cells that function to antagonize immune responses. Treg cells have the capacity to prevent potentially damaging autoimmune and protective immune responses, so the number of Treg cells is a crucial determinant of the regulatory burden on the immune system. Treg cells prevent autoimmune disease, maintain immune homeostasis, and modulate immune responses during infection. The Cloudz Human Treg Expansion Kit is designed for the robust expansion of human regulatory T (Treg) cells from CD4+ T cells. The kit contains dissolvable microspheres that are functionalized with CD3 and CD28 antibodies.
Versatility of GMP Cloudz Human Treg Cell Expansion Kit
GMP Cloudz Human Treg Cell Expansion Kit is optimized for Treg cell expansion when used in combination with
Kit Contents
• 16 mL of GMP-Grade Treg Cloudz CD3/CD28
• 100 mL of GMP-Grade Cloudz 6X Release Buffer
• Each kit contains enough reagents to activate 1x108 CD4+ T Cells
Specifications
Product Datasheets
Data Example
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Fold Expansion of Treg Cells using the GMP Cloudz Human Treg Cell Expansion Kit Primary human CD4+ cells were activated with GMP-Grade Cloudz Treg CD3/CD28 and cultured for 9 days in ExCellerate T Cell Expansion Media, Xeno-Free (R&D Systems, Catalog # CCM030) and 400 IU/mL of IL-2 (R&D systems, Catalog # 202-GMP). Expansion of FoxP3+ Treg cells was evaluated using flow cytometry on Day 5 and Day 9 and fold expansion of was calculated relative to Day 0. Error bars reflect standard deviation across 3 donors.
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Fold Expansion of CD4+/CD25+/CD127- Treg Cells using the GMP Cloudz Human Treg Cell Expansion Kit Primary human CD4+ cells were activated with GMP-Grade Cloudz Treg CD3/CD28 and cultured for 9 days in ExCellerate T Cell Expansion Media, Xeno-Free (R&D Systems, Catalog # CCM030) and 400 IU/mL of IL-2 (R&D systems, Catalog # 202-GMP). Expansion of CD4+/CD25+/CD127- Treg cells was evaluated using flow cytometry on Day 5, 7 and 9 and fold expansion of was calculated relative to Day 0. Error bars reflect standard deviation across 3 donors.
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Increased FoxP3+ CD4 Cells following Treg Expansion. Flow cytometry data showing expression of the Treg cell marker, FoxP3, in CD4+ T cells on Day 0 (A, C) and Day 9 (B, D) following expansion using the GMP Cloudz Human Treg Expansion Kit. Nine days after expansion, the cells were fixed, permeabilized, and stained with CD4-FITC, CD25-APC, and FoxP3-PE antibodies. Cells in panels A and B were stained with FoxP3 and CD4. Cells in C and D were stained with FoxP3 and CD25.
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Viability greater than 90% after 9-day expansion Viability of Human Treg after expansion with GMP Cloudz Human Treg Cell Expansion Kit. Primary human CD4+ cells were activated with GMP-Grade Cloudz Treg CD3/28 and cultured for 9 days in ExCellerate T Cell Expansion Media, Xeno-Free (R&D Systems, Catalog # CCM030) and 400 IU/mL of IL-2 (R&D systems, Catalog # 202-GMP). Cell viability after expansion for 9 days was greater than 90%. Error bars reflect standard deviation across 3 donors.
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Fold expansion of Treg Cells with GMP Cloudz Human Treg Cell Expansion Kit compared to other suppliers Fold expansion of CD25+/CD4+/FoxP3+ Treg Cells is greater with GMP Cloudz Human Treg Cell Expansion Kit compared to other GMP-Grade Treg Cell Expansion suppliers.
FAQs
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Cloudz Human T Cell Activation Kit, Catalog # CLD001, and the Cloudz Human Treg Expansion kit, Catalog # CLD006, both contain Cloudz CD3/CD28 antibodies. Can the antibodies in CLD001 be used in the CLD006 kit for Treg expansion?
While both kits have CD3/CD28antibodies, each product is manufactured and optimized to each type of cell expansion (T cell or Treg). Use of Cloudz CD3/CD28 from CLD001 in CLD006 is not supported.
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Are the Cloudz microspheres homogeneous in size?
The microspheres are not homogenous. They are a mixture of heterogeneous particles ranging in size from 8-12 um.
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Can I separate the beads from my cells with a filter rather than using the Release Buffer that is supplied with the beads?
We do not perform this type of separation is house; we would only recommend the use of the Release Buffer provided in the kit.
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Does the Release Buffer and subsequent wash step completely remove all microspheres from the cell culture?
We do not expect residual microsphers to be remaining after the Release Buffer treatment and subsequent washing step. We are in the process of developing a mass spectrometry based method to identify trace antibody and polymeric residuals in the expanded cell population. We have not visually observed any undissolved microspheres after release buffer treatment.
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Can the Cloudz microspheres be visualized under the microscope?
The Cloudz microspheres are translucent, but can be seen as faint circles underneath the layer of cells when looking at the culture through a microscope. The same brightfield setting and microscope magnification that are useful for visualizing cells can be used to visualize the microspheres.
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Has a reduction in cell number been observed after treatment of expanded cells with Release Buffer?
The Release Buffer will not have an effect on cell viability when used as recommended in the protocol. Cells should not, however, be left in release buffer for longer than 30 minutes, as that can impact viability.
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When Cloudz microspheres are added to cells in culture do they remain in suspension or do they settle down?
The Cloudz microspheres settle down along with the cells when added to culture. This is an advantage as the cells stay in close proximity to the activating reagent.
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What is the size of the Cloudz microspheres?
The Cloudz microspheres, as supplied in storage buffer, are about 2-10 µm in size. They swell to some extent in cell culture medium depending on the medium used.
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What is the concentration of Cloudz microspheres provided in this kit?
The information on concentration of microspheres (number of microsphers per volume) is proprietary. The concentration has been developed for optimal stimultaion.
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Is there informaiton available on the number of antibodies per microsphere in the Cloudz product?
The information on number of antibodies per microsphere in the Cloudz product is proprietary. The number of antibodies per microsphere has been developed for optimal performance.
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The Cloudz microspheres are not visible on the same plane as the cells when viewed under a microscpope. How do I visualize the microspheres?
The Cloudz microspheres can be seen as transluscent spheres by lowering the focal plane of the microscope. For more information, contact technical support.
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