Cellfectin

Introduction

Cellfectin® II Reagent is a proprietary, cationic lipid formulation in membrane filtered water suitable for DNA transfection into insect cells. Cellfectin® II Reagent is an improved version of the Cellfectin® Reagent and delivers the same performance as Cellfectin® with a faster protocol. Transfection using Cellfectin® II results in consistent and efficient transfection of Sf9, Sf21, and High Five™ cells in Bac-to-Bac®, BaculoDirect™, and InsectSelect™ Expression Systems from Invitrogen or equivalent systems. Cellfectin® II Reagent can also be used to transfect adherent or suspension mammalian cells. Transfection protocols for Sf9 and Sf21 cells are described in this manual. For transfecting High Five™ insect cells and mammalian cells with Cellfectin® II, refer to the Transfection Selection Tool.

Important Guidelines for Transfection

  • Cellfectin® II is a lipid suspension that may settle with time. Mix thoroughly by inverting the tube 5–10 times before use.
  • The DNA:lipid complex formation time is shorter (~15–30 minutes) when using Cellfectin® II as compared to Cellfectin® reagent.
  • Perform transfection using the appropriate protocol for the system and cell type that you are using to obtain the best results.
  • Do not add antibiotics to media during transfection as this causes cell death.
  • Transfection complexes must be formed in serum-free medium.
  • Test serum-free media for compatibility with Cellfectin® II since some serum-free formulations may inhibit cationic lipid-mediated transfection.

Optimizing Transfections

To obtain the highest transfection efficiency and minimize non-specific effects, optimize transfection conditions by varying cell density, DNA and Cellfectin® II concentrations, and transfection incubation time.

Transfecting Sf9 or Sf21 with Baculovirus DNA

Use the following procedure to transfect Sf9 or Sf21 insect cells cultured in Grace’s Insect Medium, Supplemented, containing 10% FBS in a 6-well format. All amounts and volumes are given on a per well basis.

  1. If the cell density is in the range of 1.5–2.5 × 106 cells/ml with >95% viability, and the medium is without antibiotics, proceed to step 1a. If the cell density is not in this range or the medium contains antibiotics, follow steps 1b–1c.

    • a. Add 2 ml of Grace’s Insect Medium, Unsupplemented (without antibiotics and serum) in each well. Plate 8 × 105 Sf9 or Sf21 cells per well. Do not change medium or wash the cells. The medium carried over to the next step will enhance the transfection efficiency. Allow cells to attach for 15 minutes at room temperature in the hood.
      Proceed to step 2.
    • b. Prepare 10 ml plating medium by mixing 1.5 ml Grace’s Insect Medium, Supplemented, containing 10% FBS (without antibiotics) and 8.5 ml Grace’s Insect Medium, Unsupplemented (without FBS and antibiotics).
    • c. Plate 8 × 105 Sf9 or Sf21 cells per well. Allow cells to attach for 15 minutes at room temperature in the hood. Remove the medium. Add 2.5 ml plating medium from step 1b per well. Proceed to step 2.

  2. For each transfection sample, prepare complexes as follows:

    Bac-to-Bac® SystemBaculoDirect™ System

    a. Mix Cellfectin® II before use and dilute 8 μl in 100 μl Grace’s Medium, Unsupplemented. Vortex briefly to mix.

    Note: This mixture can be kept at room temperature for up to 30 minutes


    a. Prepare Transfection Mix A: Cellfectin® II 8 μl Grace’s Insect Medium 100 μl Unsupplemented

    Note:
    This mixture can be kept at room temperature for up to 30 minutes.
     
    b. Dilute 1 μg baculovirus DNA in 100 μl Grace’s Insect Medium, Unsupplemented. Mix gently.b. Prepare Transfection Mix B: LR recombination reaction 10 μl Grace’s Insect Medium 100 μl Unsupplemented
    c. Combine the diluted DNA with diluted Cellfectin® II (total volume is ~210 μl). Mix gently and incubate for 15–30 minutes at room temperature.c. Combine Transfection mixtures A and B. Mix gently and incubate for 25–35 minutes at room temperature
  3. Add ~210 μl DNA-lipid mixture or transfection mixture (Step 2c) dropwise onto the cells from Step 1a or 1c. Incubate cells at 27°C for 3–5 hours.

  4. Remove the transfection mixture and replace with 2 ml of complete growth medium (e.g., Grace’s Insect Medium, Supplemented and 10% FBS). Use of antibiotics is optional for Bac-to-Bac® system. Use complete medium with 100 μM ganciclovir for BaculoDirect™ system.

  5. Incubate cells at 27°C for 72 hours or until you see signs of viral infection.
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Transfecting Sf9 or Sf21 with Plasmid DNA

Use this procedure to transfect Sf9 and Sf21 insect cells in a 6-well format in Sf-900 II SFM, Sf-900™ III SFM, or Grace’s Insect medium. All amounts and volumes are given on a per well basis.

  1. For log phase cells cultured in Sf-900 II SFM or Sf-900™ III SFM without antibiotics: Plate 8 × 105 Sf9 or Sf21 cells per well in 2 ml Sf-900 II SFM or Sf-900™ III SFM. Allow cells to attach for 15 minutes at room temperature.  Proceed to step 2.

     For cells cultured in of Grace’s Insect Medium: If the cell density is in range of 1.5–2.5 × 106 cells/ml with >95% viability and the medium is without antibiotics, proceed to step 1a. If the cell density is not in this range or the medium contains antibiotics, follow steps 1b–1c.

    • a. Add 2 ml of Grace’s Insect Medium, Unsupplemented (without antibiotics and serum) in each well. Plate 8 × 105 Sf9 or Sf21 cells per well. Do not change medium or wash the cells. The medium carried over to the next step will enhance the transfection efficiency. Allow cells to attach for 15 minutes at room temperature in the hood.
      Proceed to step 2.
    • b. Prepare 10ml plating medium by mixing 1.5 ml Grace’s Insect Medium, Supplemented, containing 10%FBS (without antibiotics) and 8.5 ml Grace’s Insect Medium, Unsupplemented, (without FBS and antibiotics).
    • c. Plate 8 × 105 Sf9 or Sf21 cells per well. Allow cells to attach for 15 minutes at room temperature in the hood. Remove the medium. Add 2.5 ml plating medium from step 1b per well. Proceed to step 2.


  2. For each transfection sample, prepare complexes as follows:

    • a. Dilute 1 μg plasmid DNA in 100 μl Grace’s Insect Medium, Unsupplemented (without antibiotics and serum). Vortex briefly to mix. Incubate at room temperature for 15–30 minutes.

      Optional: If you are using SFM, add 5 μl PLUS™ Reagent (Cat. no. 11514- 015) to the diluted DNA to improve transfection efficiency.

    • b. Mix Cellfectin® II before use, then dilute 8 μl Cellfectin® II in 100 μl Grace’s Insect Medium, Unsupplemented (without antibiotics and serum). Vortex briefly to mix. Incubate at room temperature for 15–30 minutes.

    • c. Combine the diluted DNA with diluted Cellfectin® II (total volume is ~210 μl). Mix gently and incubate for 5–15 minutes.


  3. Add ~210 μl DNA-lipid mixture (Step 2c) dropwise onto the cells from Step 1a or 1 c.

  4. For Sf-900 II SFM or Sf-900™ III SFM: Incubate cells at 27°C for 24–48 hours and test for transgene expression. There is no need to replace the medium. For Grace’s medium: Incubate cells at 27°C for 3–5 hours. Remove the transfection mixture and replace with 2 ml of complete growth medium (e.g., Grace’s Insect Medium, Supplemented, and 10% FBS). Addition of antibiotics to the growth medium is optional. Incubate cells at 27°C for 24–48 hours and test for transgene expression.
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100003529.pps    MAN0000759   11-Jun-2010