T Cell Activation Nanobeads，Human（RUO）

1.Precautions

1)All operations throughout the entire process must be performed in a sterile environment;

2)For initial activation, ensure that the reagent continuously interacts with the cells for 2–3 days; removing the reagent too early will result in insufficient T-cell activation and reduced proliferative capacity;

3)T cells can undergo viral transduction experiments two days after activation; the optimal transduction titer and timing should be determined independently through experimentation;

4)Residual EDTA in the cell system significantly inhibits T-cell activation stimulation; it must be thoroughly removed before starting the experiment.

2. Sample Preparation

CD4 Nanobeads, human (Cat. No. S0K0013), CD8 Nanobeads, human (Cat. No. S0K0005), and CD3 Nanobeads, human (Cat. No. S0K0011) can be used to isolate and enrich T cells from PBMCs. Refer to the corresponding product manuals for specific procedures.

Peripheral blood samples from humans need to be separated by density gradient centrifugation to obtain PBMCs, which are then used for subsequent T-cell activation experiments.

3. Cell Seeding Density and Reagent Usage Standards

This experimental protocol optimally recommends using a working dilution of 1:200; the best seeding density for sorted T cells is 1×10⁶ cells/cm²; the optimal seeding density for PBMCs is 2×10⁶ cells/cm².

Optimal Culture Medium Formulation Table for Each Type of Culture Plate

Notes: When the cell count is less than 10⁶, still add reagents in fixed volumes as per the table; when scaling up the cell culture, proportionally increase the total volumes of reagents and culture medium.

4. Standard activation procedure for a 48-well plate

1)Use AO/PI to count cells and determine the required cell amount;

2)Resuspend the cells in 995 μL of complete T-cell culture medium;

3)Add 5 μLT Cell Activation Nanobeadsand mix gently and thoroughly;

4)Place in a 37℃, 5% CO₂ incubator and let it sit for 48 hours;

5)After the initial activation, perform centrifugation and media exchange:

① Centrifuge at 300×g for 10 min;

② Slowly and completely aspirate the supernatant from the upper layer;

③ Add fresh T cell complete culture medium at a concentration of 1×10⁶/ml;

④ Incubate in a 37℃, 5% CO₂ incubator to continue culturing.

5. Cell expansion and culture procedure

1)Every 2 days, equally divide the cell suspension into new wells or flasks, and replenish with fresh culture medium;

2)Maintain standard culture conditions at 37℃ and 5% CO₂;

3)By day 12 of culture, the cells have expanded to sufficient numbers and can be used for various downstream experiments;

4)Depending on the cells' growth status and experimental design requirements, the expansion culture period can be extended up to 16 days at most;

5)If long-term expansion culture is needed, T Cell Activation Nanobeads can be added again after day 12 of culture.T Cell Activation NanobeadsPerform secondary activation stimulation.