Are the Cloudz microspheres homogeneous in size?

When in culture, the Cloudz particles are heterogeneous in size with an average diameter similar to that of a T cell.

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.

Can the Cloudz Human Treg Expansion kit, Catalog # CLD006, be used for expansion of mouse T reg cells?

We have not tested the Cloudz Human Treg expansion kit, Catalog # CLD006, with mouse Treg cells.

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.

Can the size of the microspheres provided in the Cloudz Cell Expansion products be custom designed?

Yes. Cloudz microspheres sizes can be custom made to between 2-200 µm to match your desired application. For more information, check out our Cloudz landing page: https://www.bio-techne.com/gmp-products/cloudz-cell-activation-kits. The final product would use our Custom ordering channel.

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.

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.

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.

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.

What is the composition of the Cloudz microspheres?

The Cloudz microspheres are made of alginate - a natural polymer derived from seaweed.

What is the composition of the Cloudz Release Buffer?

The composition of Cloudz Release buffer is proprietary information.

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.

What is the size of the Cloudz microspheres?

The Cloudz microspheres are 2-10 µm in supplied buffer, and quickly swell to average diameter of a T cell in cell culture media.

What is the species and source of antibodies used in the Cloudz Human Treg expansion kit, Catalog # CLD006?

The antibodies present on the microspheres supplied in CLD006 kit are monoclonal antibodies raised against human antigens and these are humanized antibodies. The monoclonal antibodies are R&D Systems' in-house clones.

What material are the Cloudz microspheres made of?

The microspheres are made of alginate co-polymer, gelled into spherical particles in the presence of divalent salt ions.

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.

Cloudz Human Treg Expansion Kit

Name: Cloudz Human Treg Expansion Kit
Brand: R&D Systems
SKU: CLD006

R&D Systems, part of Bio-Techne | Catalog # CLD006

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Product Datasheet / COA / SDS

For the robust expansion of human Treg cells from CD4+ T cells ex vivo.

Discontinued Product

CLD006 has been discontinued. View all T Cell Differentiation Kits products.

Product Summary
Preparation & Storage
Background
Product Documents
Specific Notices

Key Product Details

Species

Human

Product Summary for Cloudz Human Treg Expansion Kit

Summary

Includes dissolvable Cloudz CD3/CD28 microspheres that activate and promote the expansion of Treg cells in cell culture.

Key Benefits

Greater than 500-fold expansion of Treg cells from CD4⁺ T cells in 9 days
Cloudz CD3/CD28 microspheres are dissolvable
Easy cell harvesting using concentrated release buffer
Magnetic bead-free platform
Optimized for use with R&D Systems cytokines and media

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.

Product Details

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.

Precaution

When handling biohazardous materials such as human cells, safe laboratory procedures should be followed and protective clothing should be worn.

Limitations

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.

Formulation, Preparation, and Storage

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: T Cell Differentiation Kits

CD4+ T cells can differentiate into T helper (Th)1, Th2, Th17, and Regulatory T (Treg) cells by exposure to various cytokines and cellular interactions that induce expression of specific sets of transcription factors. Differentiation into Th1 cells is promoted through IL-12 and IFN-gamma. These cells are characterized by their secretion of IFN-gamma, IL-10, and TNF-alpha. Differentiation into Th2 cells is promoted by IL-4 in combination with either IL-2, IL-7, or TSLP. through IL-12 and IFN-gamma. These cells are characterized by their secretion of IFN-gamma, IL-10, and TNF-alpha. Th2 cells secrete IL-4, IL-5, IL-9, IL-13, and IL17E/IL-25. Differentiation into the Th17 lineage is promoted by cytokines such as TGF-beta and IL-6, while their survival and expansion are dependent on IL-21 and IL-23. Th17 cells secrete TNF-alpha, IL-6, IL-9, IL-17A, IL-17F, IL-21, IL-22, and (human) IL-26/AK155. The ability to differentiate CD4+ T cells ex vivo into Th1, Th2, Th17, or Treg cells is valuable to researchers who require specific T helper cell subsets for downstream applications, including studies for cell therapy and cancer immunotherapy.