GeticoFect Stem TransFection Reagent
Product Ordering Information
Catalog Number | Specification | Price (CNY) |
130601 | 0.75ml | 4,200.00 |
130602 | 1.5ml | 6,500.00 |
130603 | 15ml | 58,000.00 |
1. Introduction
GeticoFect Stem TransFection Reagent, developed by Getico Scientific (Shanghai) Co., Ltd., is a specialized transfection reagent designed for highly efficient delivery of nucleic acids into stem cells. It enables precise gene manipulation in stem cells, including embryonic stem cells, induced pluripotent stem cells (iPSCs), and adult stem cells. This reagent offers a reliable solution for researchers in stem cell biology, regenerative medicine, and related fields, allowing for in-depth studies on stem cell differentiation, self-renewal, and gene function.
2. Product Components
- GeticoFect Stem TransFection Reagent: The core reagent for forming complexes with nucleic acids for efficient delivery into stem cells. Supplied in a 1-ml vial.
- Storage Buffer (if applicable): Some formulations may include a buffer to maintain the stability of the reagent during storage.
3. Storage Conditions
- Store the GeticoFect Stem TransFection Reagent at 4°C. Do not freeze. Freezing can cause precipitation and inactivation of the reagent components.
- When stored under the recommended conditions, the reagent has a shelf life of 12 months from the date of manufacture.
- Avoid repeated exposure to room temperature. Return the reagent to 4°C immediately after use.
4. Pre-transfection Preparation
4.1 Cell Culture
- Cell Line Selection: The reagent is compatible with a wide range of stem cell lines. However, it is crucial to ensure that the stem cells are in a healthy, undifferentiated state before transfection. For example, if using human iPSCs, maintain them in feeder-free culture systems such as Gibco Essential 8 Medium or StemFlex Medium on defined substrates like vitronectin, Geltrex Matrix, or rh Laminin-521.
- Cell Passaging: Passage stem cells every 3-5 days, before they reach approximately 85% confluence. For routine passaging during expansion, use appropriate dissociation reagents (e.g., Gibco Versene Solution for some cell lines). When preparing cells for transfection, the dissociation method may vary depending on the cell type. For example, for some cell lines, gentle EDTA-based dissociation to generate small clumps of 3-5 cells can promote efficient transfection. For others, dissociating into a single-cell suspension using enzymes like Gibco TrypLE Select may be recommended to maximize transfection efficiency.
- Coating Culture Vessels: If required, coat the culture vessels (e.g., 6-well plates, 24-well plates, 60 cm dishes, or T-75 flasks) with the appropriate substrate. For example:
- Vitronectin Coating: Prepare a 1:100 dilution of vitronectin by diluting a 60 μl aliquot of vitronectin stock solution (0.5 mg/ml) in 6 ml of DPBS without magnesium or calcium, resulting in a working concentration of 5 μg/ml. Add 200 μl of diluted vitronectin to each well of a 24-well plate and incubate at room temperature for 1 hour to coat with 0.5 μg/cm² vitronectin. Vitronectin-coated plates can be prepared ahead of time and stored for up to 1 week at 4°C. Equilibrate at room temperature for at least 1 hour before plating cells.
- Geltrex Matrix Coating: Prepare a 1:100 dilution of Geltrex Matrix in cold Gibco DMEM/F-12 medium with GlutaMAX supplement. Add 300 μl of diluted Geltrex Matrix to each well of a 24-well plate and incubate at 37°C for ≥1 hour before use. Plates coated with Geltrex Matrix can be prepared ahead of time and stored for up to 2 weeks at 4°C. Equilibrate at room temperature for 1 hour before plating cells.
- rh Laminin-521 Coating: Prepare a 1:40 dilution of rh Laminin-521 by adding 300 μl of rh Laminin-521 stock solution (0.5 mg/ml) to 12 ml of DPBS, for a final concentration of 2.5 μg/ml. Add 400 μl of diluted rh Laminin-521 to each well of a 24-well plate and incubate at 37°C for ≥2 hours to coat the wells with 0.5 μg/cm² of rh Laminin-521. The optimal coating concentration of rh Laminin-521 can depend on the stem cell line and may range from 0.5-2 μg/cm². Increase the concentration if observing areas of incomplete cellular attachment.
4.2 Nucleic Acid Preparation
- Type of Nucleic Acids: The reagent can efficiently deliver various nucleic acids, including DNA (plasmids), RNA, siRNA, and miRNA.
- Purity and Quality: Ensure that the nucleic acid is of high purity. DNA should be free of contaminants such as proteins, lipids, carbohydrates, and endotoxins. The A₂₆₀/A₂₈₀ ratio of the DNA solution should be between 1.7 and 1.9. Prepare the DNA solution in DNase/RNase-free water or TE buffer. For RNA, ensure it is protected from degradation and is of high quality.
- Quantity: The optimal quantity of nucleic acid for transfection may vary depending on the cell type and the specific experiment. It is recommended to perform a titration experiment to determine the optimal amount. As a general guideline, start with a range of 0.5-2 μg of DNA or 50-200 nM of siRNA/miRNA per well in a 24-well plate.
5. Transfection Procedure
5.1 Complex Formation
- Reagent Equilibration: Allow the GeticoFect Stem TransFection Reagent to reach room temperature before starting the experiment.
- Dilution of Reagent and Nucleic Acid: In a sterile tube, dilute the nucleic acid (DNA, RNA, siRNA, or miRNA) in an appropriate volume of serum-free medium or Opti-MEM. In a separate tube, dilute the GeticoFect Stem TransFection Reagent in the same medium. The ratio of reagent to nucleic acid needs to be optimized for each cell type and nucleic acid type. As a starting point, for DNA transfection, a ratio of 1-3 μl of reagent per 1 μg of DNA can be tested. For RNA transfection, the ratio may be different. Mix the diluted nucleic acid and the diluted reagent gently by pipetting up and down 3-5 times.
- Incubation: Incubate the mixture at room temperature for 15-30 minutes to allow the formation of stable complexes. During this time, the reagent and nucleic acid will interact to form complexes that are ready for cell uptake.
5.2 Cell Seeding and Transfection
- Cell Seeding: Seed the stem cells in the coated culture vessels the day before transfection. The seeding density should be adjusted to ensure that the cells reach 60-80% confluence on the day of transfection. For example, in a 24-well plate, seed an appropriate number of cells (e.g., 50,000-100,000 cells per well for some stem cell lines) in complete culture medium containing serum.
- Transfection: After the complex formation, add the reagent-nucleic acid complexes directly to the wells containing the stem cells. Gently swirl the culture plate to ensure even distribution of the complexes. Do not remove the culture medium containing serum before transfection, as the reagent is compatible with serum-containing media. This simplifies the transfection protocol and reduces cell stress.
5.3 Incubation and Monitoring
- Cell Incubation: Incubate the transfected cells in a humidified incubator at 37°C with 5% CO₂. The optimal incubation time may vary depending on the experiment. For short-term studies, such as assessing transfection efficiency by fluorescence microscopy for a reporter gene, results can be observed as early as 24-48 hours post-transfection. For long-term studies, such as those involving stem cell differentiation after gene over-expression or knockdown, the cells may need to be incubated for several days or weeks.
- Cell Monitoring: Regularly monitor the cells for signs of cytotoxicity, such as cell death, changes in cell morphology, or reduced proliferation. The GeticoFect Stem TransFection Reagent has low cytotoxicity, and cell viability should remain above 90% in most cases. However, it is important to check for any adverse effects on the cells.
6. Optimization
- Reagent-Nucleic Acid Ratio: Since different stem cell lines and nucleic acid types may respond differently to the transfection reagent, it is essential to optimize the ratio of GeticoFect Stem TransFection Reagent to nucleic acid. Perform a series of experiments with varying ratios while keeping the amount of nucleic acid constant (e.g., 1 μg of DNA) and changing the volume of the reagent (e.g., 0.5 μl, 1 μl, 1.5 μl, 2 μl, 2.5 μl, 3 μl) to determine the ratio that gives the highest transfection efficiency with the lowest cytotoxicity.
- Nucleic Acid Concentration: In addition to optimizing the reagent-nucleic acid ratio, the concentration of the nucleic acid itself may need to be optimized. Test different concentrations of nucleic acid (e.g., for DNA, 0.25 μg, 0.5 μg, 1 μg, 1.5 μg, 2 μg per well in a 24-well plate) while maintaining the optimal reagent-nucleic acid ratio to find the best conditions for your specific experiment.
- Cell-Specific Optimization: Some stem cell lines may have unique characteristics that require additional optimization. For example, certain cell lines may be more sensitive to the dissociation method used before transfection, or they may respond better to transfection at a specific cell density. Adjust the cell seeding density, dissociation method, and other cell-related parameters as needed to improve transfection efficiency.
7. Troubleshooting
7.1 Low Transfection Efficiency
- Check Nucleic Acid Quality: Ensure that the nucleic acid is of high purity and integrity. Degraded or contaminated nucleic acid can lead to low transfection efficiency. Re-purify the nucleic acid if necessary.
- Optimize Reagent-Nucleic Acid Ratio: As mentioned above, re-optimize the ratio of the reagent to the nucleic acid. Try different ratios and concentrations to find the optimal conditions for your cell line and nucleic acid type.
- Cell Health and Density: Check the health of the stem cells. Unhealthy or over-confluent cells may have reduced transfection efficiency. Passage the cells at the appropriate time and ensure they are in a good physiological state before transfection. Adjust the cell seeding density if necessary.
- Complex Formation: Ensure that the reagent-nucleic acid complexes are formed correctly. Follow the incubation time and mixing procedures precisely. If the complexes are not formed properly, they may not be efficiently taken up by the cells.
7.2 High Cytotoxicity
- Reduce Reagent Concentration: If high cytotoxicity is observed, try reducing the amount of GeticoFect Stem TransFection Reagent used in the transfection. Lower the reagent-nucleic acid ratio and see if the cytotoxicity decreases while still maintaining an acceptable transfection efficiency.
- Check Incubation Time: Prolonged incubation time may increase cytotoxicity. Shorten the incubation time of the transfection complexes with the cells, for example, from the conventional overnight incubation to 4-6 hours, then replace with fresh medium and observe if the cytotoxicity decreases.
- Cell Handling: Ensure that the cells are handled gently during the transfection process. Rough handling, such as excessive pipetting or vigorous shaking, can damage the cells and increase cytotoxicity.
8. Safety Precautions
- Personal Protective Equipment: Wear appropriate personal protective equipment, including lab coats, gloves, and safety glasses, when handling the reagent.
- Avoid Ingestion and Inhalation: Do not ingest or inhale the reagent. Keep the reagent away from the mouth, nose, and eyes. In case of accidental contact with the eyes, rinse immediately with plenty of water and seek medical advice.
- Chemical Storage: Store the reagent in a secure location away from heat, light, and incompatible chemicals. Follow the storage conditions mentioned above to maintain the stability and integrity of the reagent.
- Disposal: Dispose of any unused reagent and waste materials according to local regulations. The reagent may be considered a chemical waste and should be handled appropriately.
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