In vitro Transfection Protocol

Transcription

1 BIOMOL GmbH Waidmannstr Hamburg Phone: or (D) Fax: or (D) In vitro Transfection Protocol Technical Assistance... 2 Trademarks... 2 Description and reagent selection... 3 Product information... 3 Content... 3 Regent use and limitations... 3 Formulation and Storage... 3 Quality control... 3 Reagents required Transient transfection protocol for adherent insect cells Standard protocol...4 a. Cell seeding...4 b. Transfection procedure Transfection protocol for adherent Tn5...6 a. Cell seeding...6 b. Transfection procedure Transfection protocol for insect cells grown in suspension Standard procotol...8 a. Cell Seeding...8 b. Transfection procedure in suspension Transfection for Tn5 cells grown in suspension...9 a. Cell seeding...9 b. Transfection procedure in suspension...9 Troubleshooting...10 Related reagents

2 Technical Assistance Contact the Polyplus technical support via: The Polyplus website: Phone: + 33 (0) Trademarks FectoFly, Fecturin and Polyplus-transfection are registered trademarks of Polyplus-transfection. POLYPLUS-TRANSFECTION SA Bioparc Boulevard Sébastien Brant BP ILLKIRCH cedex, FRANCE Phone : Fax: POLYPLUS-TRANSFECTION Inc Ave of the Americas, 34 th fl. NEW YORK, NY 10020, USA Phone: /11 CPT 111v A Sept 2007

3 Description and reagent selection FectoFly reagents have been developed for efficient transient transfection of insect cells and high level expression of protein or baculovirus production. is recommended for Spodoptera frugiperda (Sf21, Sf9) and Tricoplusia ni (Tn5) cell lines, while I gives higher transfection efficiency for Schneider cells (S2) derived from Drosophila melanogaster. For other cell lines derived from Bombyx mori, Helicoverpa zea, Heliothis virescens and Aniticarsia gemmatalis, we recommend testing both reagents to determine the most efficient. Product information FectoFly Kit * N 2 x 0.1 ml ml *Contains 0.1 ml, 0.1 ml I, and 10 ml 150 mm NaCl Content 1 ml of transfection reagent is sufficient to perform transfections in 6-well plates. Regent use and limitations For research use only. Not for use in humans. Formulation and Storage is provided in sterile apyrogenic water. FectoFly reagents are shipped and stored at 4 C. Do not freeze. At 4 C, is stable for 1 year. Quality control Each batch of is tested in a transfection assay using insect cells. Reagents required Cell culture medium is required to dilute reagent and DNA for Sf21, Sf9 and Tn5 cells. 3/11 CPT 111v A Sept 2007

4 1. Transient transfection protocol for adherent insect cells 1.1 Standard protocol The protocol is adapted for standard cell lines such Sf9, Sf21. For Tn5 and derivatives see section 1.2. a. Cell seeding The cell density is a critical parameter for efficient insect cells transfection. In order to achieve optimal transfection efficiency with, we recommend preparing a cell suspension at 1.25 x 10 6 cells/ml. The volume to dispense per well is shown in Table 1. Table 1. Recommended volume of cell suspension to seed 2 h before transfection Culture vessel Volume of cell suspension to add per well Corresponding number of cells per well 96-well 80 µl 1 x well 400 µl 5 x well/35 mm 1.6 ml 2 x cm 4 ml 5 x 10 6 b. Transfection procedure We recommend using a 1:1 ratio DNA to (w/v). 1 µg of DNA per µl of 4/11 CPT 111v A Sept 2007

5 Protocol for 6-well plates using Per well in 6 well plates: Seed 1.6 ml of a cell suspension at 1.25 x 10 6 cells/ml. Allow cells to attach for 2 h in the incubator. Dilute 9 µg DNA into 100 µl insect cell culture medium (without serum), mix by pipetting up and down or by vortexing. Dilute 9 µl into 100 µl insect cell culture medium (without serum), mix. Add 100 µl to 100 µl DNA (in this order), vortex for 10 s and incubate for 30 min at RT. Add 200 µl FectoFly I/DNA complexes dropwise to the cells to distribute the complexes evenly. Gently rock the plates back and forth and from side to side. Return the plates to the incubator for 4 h. Add 3ml of medium with serum (and antibiotics/antimycotics if required). Proceed with protein purification after 72 h or as required.* For other culture formats, apply the volumes and amounts indicated in Table 2 to the 6-well plates protocol above. 5/11 CPT 111v A Sept 2007

6 Table 2. Amount of DNA and volumes of reagent to be used for transfection of most insect cells according to the cell culture formats. Culture vessel Amount of DNA (µg) Volume of 1:1 ratio Range of for optimisation Volume of dilution buffer for DNA and Total volume of complexes added per well Volume of medium to add after 4 h 96-well µl 24-well µl 6-well ml 10 cm ml If optimisation is required in 6-well plates, test 6 to 9 µg of DNA. 1.2 Transfection protocol for adherent Tn5 This protocol is suitable for larger insect cells such as Tn5 and derivatives. a. Cell seeding The cell density is a critical parameter for efficient insect cells transfection. In order to achieve optimal transfection efficiency with and Tn5 cells, we recommend preparing a cell suspension at 5 x 10 5 cells/ml. The volume to dispense per well is shown in Table 3. Table 3. Recommended volume of cell suspension to seed 2 h before transfection Culture vessel Volume of cells suspension to add per well Corresponding number of cells per well 96-well 80 µl 4 x well 400 µl 2 x well/35 mm 1.6 ml 8 x cm 4 ml 2 x /11 CPT 111v A Sept 2007

7 b. Transfection procedure We recommend using a 1:1 ratio DNA to (w/v). 1 µg of DNA per µl of Per well in 6-well plates: Seed 1.6 ml of a cell suspension at cell/ml. Allow cells to attach for 2 h in the incubator. Dilute 6 µg DNA into 100 µl insect cell culture medium (without serum), mix by pipetting up and down or by vortexing. Dilute 6 µl into 100 µl insect cell culture medium (without serum), mix. Add 100 µl to 100 µl DNA (in this order), vortex for 10 s and incubate for 30 min at RT. Add 200 µl /DNA complexes dropwise to the cells in order to distribute the complexes evenly. Gently rock the plates back and forth and from side to side. Return the plates to the incubator for 4 h. Add 3 ml of complete medium containing serum (and antibiotics/antimycotics if required). Proceed with protein purification after 72 h or as required. For other culture formats, apply the volumes and amounts indicated in Table 4 to the 6- well plates protocol above. Table 4. Amount of DNA and reagent to use for transfection of Tn5 cells according to the cell culture format. Culture vessel Amount of DNA for Tn5 (µg) Volume of 1:1 ratio Range of for optimization 96-well well well / 35 mm cm /11 CPT 111v A Sept 2007

8 2. Transfection protocol for insect cells grown in suspension 2.1 Standard procotol The protocol is adapted for standard cell lines such Sf9, Sf21 grown in suspension. For Tn5 and derivatives see section 2.2. a. Cell Seeding The cell density is a critical parameter for efficient insect cells transfection. In order to achieve optimal transfection efficiency with, we recommend preparing a cell suspension at 10 6 cells/ml in synthetic medium and antibiotics if needed. Keep under agitation at 50 to 100 rpm. b. Transfection procedure in suspension We recommend using a 1:1 ratio DNA to (w/v). Per ml of culture, use 2 µg of DNA and 2 µl of Protocol for 25 ml cell culture using 8/11 CPT 111v A Sept 2007

9 Per ml of cell culture volume: Seed the required volume of cell suspension at 10 6 cells/ml. Keep under agitation. Dilute the DNA in synthetic medium as indicated in Table 5, mix by pipetting up and down or by vortexing. Dilute in synthetic culture medium as indicated in Table 5, mix. Add to the DNA, vortex for 10 s and incubate for 30 min at RT. Add the /DNA complexes to the cells. Incubate for 24 to 72 h under agitation in standard cell culture conditions. Proceed with protein purification as required. Table 5. Volume of complexes to prepare according to the chosen volume of culture Volume of cell suspension Size of cell culture vessel Volume of cell culture medium for both DNA and FectoFly TM I Volume of complexes added to the cells 3 ml 50 ml tube 100 µl 200 µl 25 ml 250 ml erlenmeyer 1 ml 2 ml 50 ml 250 ml erlenmeyer 2 ml 4 ml 1 L bioreactor 40 ml 80 ml 2.2 Transfection for Tn5 cells grown in suspension This protocol is suitable for other large insect cells such as Tn5 and derivatives. a. Cell seeding The cell density is a critical parameter for efficient insect cells transfection. In order to achieve optimal transfection efficiency with in Tn5 cells, we recommend preparing a cell suspension at 3 x 10 5 cells/ml in synthetic medium containing antibiotics. Keep under agitation at 50 to 100 rpm. b. Transfection procedure in suspension Proceed as in the standard protocol described in section 2.1.b using as before 2 µg of DNA and 2 µl of per ml of culture. 9/11 CPT 111v A Sept 2007

10 Troubleshooting Observations Comments and Suggestions Low transfection efficiency Low protein yields Optimize the amount of plasmid DNA used in the transfection assay by testing a various amounts of DNA. Use high quality plasmid preparation, free of RNA (the OD260/280 ratio should be greater than 1.8) Check the agitation used for cell culture ( rpm) Optimize the /DNA ratio from 1:1 to 1:2 Perform a positive control transfection experiment with a well-characterised reporter gene (Luciferase or GFP expressed from commercially available plasmid) Use 150 mm NaCl as a dilution buffer Try I Seed less cells per well Check culture conditions and passage cells at lower density Try a different culture medium Check the toxicity of the protein Related reagents Fectofly II for transfection of other insect cells such as Schneider cells (S2). Fecturin for protein and virus production in mammalian cells grown in synthetic media. 10/11 CPT 111v A Sept 2007

11 NOTES BIOMOL GmbH Waidmannstr Hamburg Phone: or (D) Fax: or (D) 11/11 CPT 111v A Sept 2007