Flow Cytometry FAQs

1. If the trypsin digestion process alters the antigenic epitopes, it may impact subsequent detection results. 2. During continuous culturing, cell lines undergo constant mutations. Prolonged and repeated passaging may lead to changes in both the genotype and phenotype of the cell line.

Standardized washing procedures with magnetic bead buffer generally do not negatively impact subsequent cell culture viability. In fact, they can help improve cell activity and cultivation outcomes. The magnetic bead storage solution contains stabilizers (such as BSA or FBS) but may also include components that are unfavorable for cell culture (e.g., sodium azide). The purpose of washing is to remove these storage solution components and avoid their potential cytotoxic effects.

1. The size of CD3/CD28 magnetic beads is similar to that of natural antigen-presenting cells (APCs), making their activation mechanism closer to that of natural APCs. They can form immune synapses, and this physical cross-linking method efficiently activates T cells with strong and uniform signals. In contrast, CD3/CD28 purified antibody stimulation relies on the distribution of soluble antibodies, which struggle to form effective cross-linking signals, resulting in weaker signal strength. Although plate-coated antibodies can provide cross-linking, signal distribution may be uneven. 2. CD3/CD28 magnetic beads are relatively simple to operate, as they do not require coating and can be directly added to cell suspensions while being easy to remove. On the other hand, CD3/CD28 purified antibody stimulation often requires coating on culture plates, involves more operational steps, and the antibodies are difficult to remove. 3. CD3/CD28 magnetic beads are more conducive to preserving naive and memory T cell phenotypes, with a lower degree of T cell exhaustion and longer-lasting efficacy. In contrast, CD3/CD28 purified antibody stimulation tends to lead to terminal differentiation, potentially resulting in a higher proportion of effector T cells and a relatively higher risk of exhaustion. 4. The initial cost of CD3/CD28 magnetic beads is higher, while the cost of CD3/CD28 purified antibodies is relatively lower.

The recommended ratio of magnetic beads to T cells is 1:1. With a bead concentration of 1×10^8 beads/mL, 1 mL of the product can stimulate and activate 1×10^8 cells.

Individual variations in blood composition, sample preparation, and experimental operations significantly affect T cell purity. For normal human peripheral blood samples, under optimal operating conditions, approximately 2×10^6 to 3.5×10^6 T cells can be isolated from 5 mL of blood.