OptiLNP RNA Transfection Reagent (Primary Immune Cells) 

Lipid nanoparticles (LNP) are a lipid-based nanoscale drug delivery system widely used for the delivery of nucleic acids (such as mRNA, siRNA, DNA, etc.) and small molecule drugs.

Naked RNA is a negatively charged hydrophilic macromolecule that faces challenges in entering cells due to electrostatic repulsion from the cell membrane and is easily degraded by RNases. By encapsulating RNA within LNP, RNA can effectively traverse the cell membrane and be released into the cytoplasm, enabling efficient delivery.

MCE OptiLNP RNA Transfection Reagent (Primary Immune Cells) is a ready-to-use transfection reagent based on LNP technology. It utilizes the unique transmembrane transport and escape mechanism of LNP to achieve efficient delivery of RNA, making it suitable for transfection of various types of RNA in Primary Immune Cells.

Features of MCE OptiLNP RNA Transfection Reagent (Primary Immune Cells)

1. Lipid Nanoparticle (LNP) Technology: Utilizes LNP carriers to effectively protect RNA from degradation and immune system clearance during the delivery process.

2. Efficient Intracellular Delivery: The unique transmembrane transport and escape mechanisms of LNPs achieve an encapsulation rate of over 80% for small sample amounts, enabling efficient transfection of RNA in primary immune cells.

3. Wide Applicability: Suitable for in vitro RNA transfection of various types of primary immune cells. Supports a variety of RNA types such as siRNA, miRNA, gRNA and mRNA.

4. User-Friendly Operation: No encapsulation equipment is required; RNA-LNP encapsulation and transfection can be completed in a single mixing step, and there is no need to replace with fresh culture medium after transfection.

5. Biocompatibility: Low cytotoxicity, effectively balancing high transfection efficiency with cell viability, and providing gentle action.

6. Safety: The formulation uses biodegradable lipid materials to ensure safety.

4℃, 1 year

Do not freeze.

1. This product has been filtered using a 0.22 μm membrane.

2. The transfection enhancer is provided as a lyophilized powder. Before opening the vial, please centrifuge.

3. For optimal transfection efficiency, it is recommended to dissolve the RNA stock in nuclease-free sterile water. Other solvents may affect transfection efficiency.

4. Recommended RNA stock concentration: mRNA > 200 ng/μL, Short-chain RNA > 100 ng/μL, siRNA > 10 μM.

5. It is recommended to prepare RNA pre-mixes using the dilution buffer provided with the kit to dilute the RNA stock solution.

6. The recommended concentration for RNA premix is 40 ng/μL. For co-transfection of multiple RNAs, the RNA premix concentration should be the sum of the concentrations of all RNAs. For example, in co-transfection of CRISPR Cas9 mRNA and gRNA, the RNA pre-mix concentration should be the sum of the Cas9 mRNA and gRNA concentrations. The recommended ratio of Cas9 mRNA (μg) to gRNA (μg) is 1:1 - 1:2.

7. Common short-chain RNAs (e.g., siRNA) typically weigh between 13 - 15 μg per 1 nmol without special modifications. The exact weight can be calculated based on the specific nucleic acid sequence and modifications.

8. The RNA/LNP ratio is crucial for LNP encapsulation. If the per-well RNA amount needs adjustment, the volume of RNA-LNP complex added to each well should be proportionally adjusted.

9. RNA-LNP complexes can be stably stored at room temperature for up to 8 h. Do not store RNA-LNP complexes under freezing conditions.

10. There is no need to replace the culture medium before or after transfection. Complete medium containing serum and antibiotics does not significantly affect transfection efficiency.

11. 6 h after transfection, standard cell culture processes, such as medium replacement, can be performed.

12. Cell conditions greatly influence transfection efficiency. It is recommended to use cells in good growth co

Human Primary T Cell Activation and Culture Reference Protocol

1. Cell Thawing

1) Thaw the human primary T cell cryovial in a warm water bath for cell recovery. Alternatively, human primary T cells isolated from peripheral blood can be used directly.

2) Add complete culture medium and place the cells in the incubator for culture.

Note: a. Both serum-free or serum-containing culture media can be used for primary T cell culture.

b. It is recommended to perform activation treatment on the isolated or thawed T cells immediately.

2. Cell Activation and Expansion Culture

Count the T cells add an appropriate amount of human CD3/CD28 T Cell Activation Magnetic Beads (MCE Catalog No. HY-K0353) for activation and expansion culture.

Note: a. T cell activation can also be performed using T cell activators.

b. For magnetic bead-based T cell activation, refer to the instructions provided with the T Cell Activation Magnetic Beads.

c. The duration of T cell expansion can be adjusted based on the cell quantity, but it is generally recommended to perform activation for 3 days before proceeding.

d. During cell culture, medium replenishment or partial medium change can be performed as needed.

3. Expression Level Detection

Use flow cytometry to detect the surface expression level of CD25 on activated T cells. Transfection experiments can be performed when CD25 expression exceeds 80%.

Note: a. For 24-well plates, it is recommended to use a cell density of 2 - 5 × 10⁵ cells per well. For other culture plate formats, adjust the cell density according to standard experimental protocols.

b. It is recommended to complete RNA transfection experiments within one week after T cell activation.

1. Prepare RNA Pre-mix Solution: Dilute the RNA stock solution with the dilution buffer. Adjust the RNA concentration to 40 ng/μL or the siRNA concentration to 3 pmol/μL.

2. Prepare RNA-LNP Complex: Mix equal volumes of transfection reagent and RNA pre-mix directly. Pipette up and down at least 20 times to ensure thorough mixing.

3. Preparation of Transfection Enhancer Solution: Dissolve the Transfection Enhancer (Lyophilized) in PBS (1×) to prepare a transfection enhancer solution with a concentration of 1 μg/μL.

Note: Note: The transfection enhancer solution can be stored at -20°C for 3 months, it is recommended to avoid repeated freeze-thaw cycles.

Refer to Tables 1 and 2. Add RNA-LNP Complex and Transfection Enhancer Solution to each well, mix gently and incubator for further culture.

After incubation of 24 - 48 h, the transfection effect can be analyzed by fluorescence detection, Western Blot, ELISA, flow cytometry, reporter gene, immunofluorescence staining and other methods according to the experimental needs.

1. This product has been filtered using a 0.22 μm membrane.

2. The transfection enhancer is provided as a lyophilized powder. Before opening the vial, please centrifuge.

3. For optimal transfection efficiency, it is recommended to dissolve the RNA stock in nuclease-free sterile water. Other solvents may affect transfection efficiency.

4. Recommended RNA stock concentration: mRNA > 200 ng/μL, Short-chain RNA > 100 ng/μL, siRNA > 10 μM.

5. It is recommended to prepare RNA pre-mixes using the dilution buffer provided with the kit to dilute the RNA stock solution.

6. The recommended concentration for RNA premix is 40 ng/μL. For co-transfection of multiple RNAs, the RNA premix concentration should be the sum of the concentrations of all RNAs. For example, in co-transfection of CRISPR Cas9 mRNA and gRNA, the RNA pre-mix concentration should be the sum of the Cas9 mRNA and gRNA concentrations. The recommended ratio of Cas9 mRNA (μg) to gRNA (μg) is 1:1 - 1:2.

7. Common short-chain RNAs (e.g., siRNA) typically weigh between 13 - 15 μg per 1 nmol without special modifications. The exact weight can be calculated based on the specific nucleic acid sequence and modifications.

8. The RNA/LNP ratio is crucial for LNP encapsulation. If the per-well RNA amount needs adjustment, the volume of RNA-LNP complex added to each well should be proportionally adjusted.

9. RNA-LNP complexes can be stably stored at room temperature for up to 8 h. Do not store RNA-LNP complexes under freezing conditions.

10. There is no need to replace the culture medium before or after transfection. Complete medium containing serum and antibiotics does not significantly affect transfection efficiency.

11. 6 h after transfection, standard cell culture processes, such as medium replacement, can be performed.

12. Cell conditions greatly influence transfection efficiency. It is recommended to use cells in good growth co