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CAR T cell research

Enrichment of CD4⁺ and CD8⁺ T cells by positive selection using a mixture of CD4 and CD8 MicroBeads (1:1) in combination with the MACS Technology, either manually or automatically using the autoMACS® Pro. Cells were fluorescently stained with CD45-VioBlue®, CD4-VioGreen™, and CD8-APC-Vio® 770 and analyzed by flow cytometry using the MACSQuant® Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and 7-AAD fluorescence.

Human T cells were isolated using the Pan T Cell Isolation Kit, human. Purified T cells were stimulated with T Cell TransAct, human and cultured in TexMACS Medium containing IL-2. CD4, CD8, CD25, and CD69 expressions were analyzed via flow cytometry on a MACSQuant® Analyzer. Proliferation was measured using the cell division tracker CFSE. T cells activated with T Cell TransAct, human showed high expression of activation markers CD25 and CD69 after 48 h, as well as strong proliferation after 7 days of culture. 

Human T cells were transduced with gamma-retroviral vectors encoding GFP in the presence of Vectofusin-1. Transduction was performed either at 37 °C in an incubator (static), or the T cells were centrifuged at 400×g for 2 hours at 32 °C (spin) before they were transferred to 37 °C. GFP expression was analyzed on day 7 of cultivation using the MACSQuant^®Analyzer. T cell viability was assessed by flow cytometry using Viobility™ Fixable Dye.

Human T cells were isolated from peripheral blood mononuclear cells (PBMCs) using the Pan T Cell Isolation Kit, human, and stimulated with the T Cell Activation/Expansion Kit, human. Cells were cultivated in the presence of IL-2 for 14 days in either TexMACS GMP Medium, TexMACS Medium, research grade, a competitor’s T cell medium, or basal medium (RPMI + 10% serum). CD4 and CD8 expressions were analyzed via flow cytometry on a MACSQuant® Analyzer and cell expansion was evaluated by cell counting at culture onset and after 14 days of culture. Highest expansion rates were achieved when cultivating T cells in TexMACS Medium (MACS GMP and research grade) compared to a competitor’s T cell medium or basal medium.

Activity of Human IL-2 IS, premium grade, (dark purple bar) was compared to another commercially available product (light purple bar).

After single beads are identified, color-coded MACSPlex bead populations are automatically identified by their fluorescence properties in the PE and FITC channel. Standard curves for each investigated cytokine in the unknown sample are generated. The median of the APC fluorescence of each captured bead population gives the concentration of each cytokine in the unknown sample. Results are displayed as a graph and as absolute numbers.