Background

    Dendritic cells (DCs) are named as dendritic cells because their cell membranes extend outwards to form membranous dendrites similar to nerve cell axons. DCs are the most powerful professional antigen presenting cells (APCs) in the body, which can efficiently take up, process and present antigens to stimulate the activation of naive T cells. 

    This product is suitable for the expansion and culture of human peripheral blood-derived dendritic cells and can obtain high-viability and high-purity DCs. This kit is limited to in vitro research use.

NKT cell activation and expansion reagent

【Notes】

1. Before use, the reagents in the kit need to be centrifuged briefly at low speed (e.g. 1000g/min for 1min) to ensure that the reagent volume is accurate;

2. During the culture process, DC cells gradually transition from adherent growth to a semi-suspended or fully suspended state. DC cells are a non-proliferating cell population. It is recommended to conduct subsequent experiments as soon as possible after maturity.

3. Due to variability between samples, the cell induction rate may vary from sample to sample;

4. Most of the DC cells induced by this kit are in a suspended state, and a small number exist in an adherent state. The adherent or suspended state of the cells may vary due to differences between samples.

Human Dendritic Cell Expansion Culture Kit Operation Procedure

Day 0 (PBMC isolation and seeding)：

1. Mononuclear cell layer separation: Take a small amount of blood sample from 50-60 mL of fresh heparin anticoagulated whole blood from the donor for aseptic streak test and use lymphocyte separation fluid or lymphocyte separation tube to separate mononuclear cells from the remaining blood sample (see the instructions for use of the corresponding product for details). After centrifugation, cell stratification can be seen, with the bottom layer being red blood cells, the top layer being plasma, and the middle white film layer being the mononuclear cell layer.

2. Preparation of heat-inactivated autologous plasma: draw the upper plasma (be careful to avoid contact with the buffy coat) into a centrifuge tube and centrifuge at 1500 g for 10 min at room temperature. Transfer the supernatant to a new centrifuge tube and inactivate at 56°C for 30 min, then cool in a 4°C refrigerator for 30 min (or place in a -20°C refrigerator for 15 min), invert and mix 1-2 times, then centrifuge at 1500 g for 10 min at room temperature, and transfer the supernatant to a new centrifuge tube to obtain heat-inactivated autologous plasma.

3. Washing of mononuclear cells: Pipette the buffy coat layer (mononuclear cell layer) into a 50 mL centrifuge tube, add PBS/DPBS or 0.9% saline without calcium or magnesium (1-2% 20% human albumin can be added), mix well, centrifuge at 300-500 g for 10 min at room temperature, and wash 1-2 times. Resuspend the cells in 30 mL DC serum-free medium, plate them in a TC-treated T75 culture flask, and incubate them flat in a 37°C, 5.0% CO 2 incubator for 2-2.5 h.

4. Transfer of suspended cells: After incubation for 2 to 2.5 hours, gently shake the T75 culture flask, transfer the suspended cells to another new culture flask, and continue to culture CIK/NKT cells as needed (it is recommended to use Anage- NKT Cell Activation and Expansion Culture Kit, catalog number DNK2308).

5. DC cell culture: Gently wash the original T75 flask twice with 15-20 mL of normal saline or PBS/DPBS without calcium and magnesium, then add 27 mL of DC serum-free culture medium, 3 ml of heat-inactivated autologous plasma and one tube of DC-A factor to a total volume of 30 mL and continue to culture in a 37°C, 5.0% CO2 incubator .

Day 3 (3×24h): Replenish reagents：

Add one tube of DC-B factor to the T75 culture flask in culture, shake gently, and continue to culture in a 37°C, 5% CO 2 incubator.

Day 5: Replenish reagents

Add a DC-C factor to the T75 culture flask in culture. If DC cells need to be loaded with antigens, the corresponding specific antigen should be added at the same time.

Day 7: Harvest cells：

1. Usually, DC cells gradually change from adherent growth to semi-suspension/suspension growth. DC cells are a non-proliferating cell population. It is recommended to conduct subsequent experiments as soon as possible after the cells mature.

For example, mature DC cells can be collected and mixed with the NKT/CIK cells cultured to Day 7 to perform DC-NKT/CIK co-culture.

2. Most mature DC cells are in a suspended state with detached cells. Dendrite-like protrusions can be seen on the cell surface. The shapes are irregular, with different lengths and thicknesses. The cells are large and transparent. The morphological light microscopic photograph of DC cells is shown in the figure below (200×).

DC Quality Inspection：

1. Proportion of living cells: Tested by trypan blue staining, the proportion of living cells should be above 90%;

2. Morphological observation: more than 90% of the cells were in a semi-suspended state, and the cells had multiple antennae-like protrusions;

3. Cell phenotype analysis (see the figure below): On the 7th Day of culture, the cells were collected and sampled, and the expression of molecules such as CD80, CD83, CD86, CD40, HLA-DR and CD14 on the surface of DC cells was detected by flow cytometry. Mature DC usually showed low expression of CD14 (<5%), while other molecules were highly expressed. CD83 is a specific marker for mature DC, which is not expressed or expressed at a low level on the surface of monocytes and immature DC.