Pan T Cells Cytotoxicity Assay

Human Pan T cell Isolation

The human Pan T cells may be isolated from mononuclear cells using an indirect immunomagnetic method that utilizes antibodies against the cell surface marker for monocytes, neutrophils, eosinophils, B cells, stem cells, dendritic cells, NK cells, granulocytes, or erythroid.  

The cocktail includes antibodies against cells markers CD14, CD16, CD19, CD34, CD36, CD56, CD123, CD235a. 

General Protocol for Pan T cell Isolation (Miltenyl Biotec)

Pan T Cell Biotin-Antibody Cocktail, human: Cocktail of biotin-conjugated monoclonal antibodies against CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a (GlycophorinA).

MicroBeads conjugated to monoclonal anti-biotin antibody (isotype: mouse IgG1) and monoclonal anti-CD61 antibody (isotype: mouse IgG1).

Prepare cells and determine cell numbers.

• Resuspend cell pellet in 40 μL of buffer per 10⁷ total cells.
• Add 10 μL of Pan T Cell Biotin-Antibody Cocktail per 10⁷ total cells.
• Mix well and incubate for 5 minutes in the refrigerator (2−8 °C).
• Add 30 μL of buffer per 10⁷ total cells.
• Add 20 μL of Pan T Cell MicroBead Cocktail per 10⁷ total cells.
• Mix well and incubate for 10 minutes in the refrigerator (2−8 °C).
• Proceed to subsequent magnetic cell separation.
• Place LS Column in the magnetic field of a suitable MACS Separator. For details refer to the respective MACS Column datasheet.
• Prepare column by rinsing with 3 mL of buffer.
• Apply cell suspension onto the column. Collect flow-through containing unlabeled cells, representing the enriched T cells.

Flow Cytometric Analysis & Identification of T cells using cell surface markers

• Presence of CD3+, CD2+
• Presene of CD3+, CD44-, CD62+

Co-Incubation of T cells (Effector cells), Tumor cells (Target) & MAb/ Bispecific antibodies/ADCs

• Culture T cells isolated from humans and target tumor cells separately. The tumor cells depend on the target of MAb/ Bispecific antibodies/ADCs. Tumor cells lines include Raji cells (CD19), SKOV3 (Ovarian Cancer), SKBR3 (Breast cancer, HER2+), SW480 ( EGFR+)
• Transfer Target cells (2500) with T cells (25,000) at 1:10 Target to Effector ratio to the RPMI media containing 10mM HEPES, 50 uM beta-mercaptoethanol, 1 mM sodium pyruvate, 100 U/ mL Penicillin. 
• Add different concentrations of  MAb/ Bispecific antibodies/ADCs for stimulation and cytotoxicity testing. 
• Incubate the mixture for 48 hours 
• Use the assay kit that utilizes the presence of ATP such as Luciferin Cell Glo Titer or LDH method for cell viability.