Cloudz Human T Cell Activation Kit

FAQs for Cloudz Human T Cell Activation Kit

• When total PBMCs are used for expansion in the GMP Cloudz Human T Cell Activation Kit, catalog # CLD001, does the presence of other cell types effect the degree of T-cell expansion?

  We have used catalog # CLD001 with PBMCs and found that the presence of other cells does not interfere with the expansion of CD3+ cells. Normally, at the end of the 9-day culture we see greater than 95% CD3+ cells. 

• 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. 

• What seeding density is optimal for the Cloudz Human T Cell Activation Kit, Catalog #  CLD001?

  A seeding cell density of 0.25 x 10⁶ cells/mL is a good starting density. However, further optimization may be required as there will be donor-to-donor variation.

• 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. 

• 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.

•  Does Cloudz™ Human T Cell Activation Kit, catalog # CLD001, support activation of pure pan T cells or PBMCs?

  The Cloudz™ Human T Cell Activation Kit is designed to activate and expand human T cells
  from peripheral blood mononuclear cells (PBMCs) or from enriched T cell populations. 

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

• 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.

• When using Cloudz Human T Cell Activation Kit, catalog # CLD001, for T cell expansion, are the microsphere bound antibodies attached to cells at the end of culture?

  When using our Cloudz CLD001 microspheres for T cell expansion, the microspheres are dissolved (using release buffer) after expansion. The antibodies on the microspheres are also removed on spinning the expanded cells down.

• 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 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.

• Can Cloudz Human T cell activation kit, Catalog # CLD001, be used for single cell cloning experiments?

  We have not evaluated the use of the Cloudz Human T cell Activation kit in single cell cloning experiments.