Why Expand Tregs Ex Vivo Using Cloudz Human Treg Expansion Kit?
Forkhead Box P3 (FoxP3)+ regulatory T (Treg) cells are a suppressive subset of CD4+ T cells that function to antagonize immune responses. Treg cells play an important role for preventing autoimmune disease, maintaining immune homeostasis, and modulating immune responses during infection. In vitro differentiation of Treg cells from the larger naïve CD4+ T cell population is often used to obtain large numbers of Tregs for downstream experimentation. Efficient methods of Treg expansion that enable easy cell harvesting are necessary to facilitate basic discovery research and to elucidate their application in preclinical and therapeutic settings.
The Cloudz Human Treg Expansion Kit is designed for the robust expansion of human regulatory T cells (Tregs) cells from CD4+ T cells. The kit contains dissolvable microspheres that are functionalized with anti-CD3 and anti-CD28 antibodies (Cloudz Treg CD3/CD28). The kit also contains Cloudz 6X Release Buffer which quickly dissolve Cloudz Treg CD3/CD28, enabling easy cell harvesting for downstream processing. Each kit contains enough materials to activate and expand FoxP3+ Tregs from 1 x 108 CD4+ T cells, with greater than 500-fold expansion in 9 days.
How to Optimize Treg Expansion using the Cloudz Human Treg Expansion Kit?
This kit was designed as a versatile expansion reagent to support Treg cell expansion in vitro in combination with a variety of media and cytokine combinations. The datasheet contains an optimized protocol for expansion using Recombinant Human IL-2 and ExCellerate Human T Cell Expansion Media. Using the recommended protocol Treg cell can achieve greater than 500-fold expansion in 9 days.
If other cytokines, cytokine concentrations, or media are desired, the expansion protocol may need to be optimized from the provided protocol in the datasheet.
16 mL of Cloudz Treg CD3/CD28
100 mL of Cloudz 6X Release Buffer
Stability and Storage
Upon receipt, this media should be stored at 2-8 °C. Do not use past kit expiration date.
When handling biohazardous materials such as human cells, safe laboratory procedures should be followed and protective clothing should be worn.
FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
The safety and efficacy of this product in diagnostic or other clinical uses has not been established.
This reagent should not be used beyond the expiration date indicated on the label.
Results may vary due to variations among cells derived from different donors.
Fold Expansion of Treg Cells using the Cloudz Human Treg Expansion Kit. Human CD4+ T cells were cultured for 9 days following the protocol and using reagents included in the Cloudz Human Treg Expansion Kit. Expansion of FoxP3-positive Treg cells was evaluated using flow cytometry on Day 5 and Day 9 and fold expansion of was calculated.
Increased FoxP3-positive CD4+ T Cells Following Treg Cell Expansion. Flow cytometry data showing expression of the Treg cell marker, FoxP3, in CD4+T cells on Day 0 (A,C) and Day 9 (C, D) following expansion using the 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.
Preparation & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Currently there are no citations for Cloudz Human Treg Expansion Kit.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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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