UltraBio™ DNA Transfection Reagent for Insect Cell

Aladdin's UltraBio™Insect Transfection Reagent is a novel, cationic liposome-mediated transfection formulation developed for highly efficient transfection of DNA into insect cells, with good consistency and extremely low cytotoxicity. It can be used to transfect baculovirus plasmid (bacmid) DNA, such as pFastBac1, into insect cells such as Sf9, Sf12, and High Five™, facilitating the expression of target genes with Bac-to-Bac®, BaculoDirect™, and InsectSelect™ expression systems.UltraBio™Insect Transfection Reagent is optimized specifically for high-performance transfection of insect cells, with a transfection efficiency of over 98% in Sf9 cells (Figure 1). Please note that the conventional liposome transfection reagents, such as UltraBio™6000 transfection Reagent and UltraBio™2000 Reagent, are unable to transfect insect cells effectively.Figure 1. Sf9 insect cells transfected with EGFP recombinant bacmid using Aladdin's UltraBio™Insect Transfection Reagent . pFastBac-GFP plasmid was transformed into DH10Bac strain to obtain EGFP recombinant bacmid which was then transfected into Sf9 insect cells. Cells were harvested and observed in the bright field (A) and the fluorescence channel (B) under fluorescence microscopy 96 hours after transfection. UltraBio™Insect Transfection Reagent is easy to use. Its transfection protocol is the same as the commonly used Cellfectin® II Reagent.Generally, it takes at least 96 hours after transfection of expression plasmid to yield high protein expression levels in insect cells.The transfection efficiency can be determined by transfecting bacmid carrying genes encoding fluorescent proteins, such as EGFP.For insect cells cultured in 6-well plates, C0551-0.5ml is sufficient for 60 transfections, C0551-1.5ml is sufficient for 180 transfections, and C0551-7.5ml is sufficient for 900 transfections.Precautions：Use high-quality DNA to achieve higher transfection efficiency.Make sure that the cells are actively dividing and reach the appropriate density at the time of transfection.Serum-free insect culture medium without antibiotics is required, but not supplied in the product. SFX-INSECT (SH30278, Hyclone), Grace's Insect Medium, Unsupplemented (11595030, Gibco), or Grace's Insect Medium, Supplemented (11605094, Gibco) can be purchased separately.Mix LipoInsect™ Transfection Reagent gently before using. Vortex or centrifuge should be avoided.Cover the LipoInsect™ Transfection Reagent tightly after each use to avoid long time exposure to air that may reduce the transfection efficiency.This product is hazardous. Please take effective measures to avoid direct contact or inhalation.This kit is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1. Bacmid transfection:The following procedures are for transfecting bacmid into insect cells in a 6-well plate. Alternative culture vessels require adjustment of the amount of Transfection Reagent and cell cultures as indicated in the table below.a. Plate insect cells in 6-well plates:Plate approximately 8×105 Sf9 or Sf21 cells in a complete growth medium per well (Cell number plated is dependent on the cell type, the size and growth rate of cells). Incubate cell cultures at 27-28ºC. The following procedures for transfection and culturing are all performed at this temperature.b. When cell density reaches 70-90%, replace the complete growth medium with 0.8ml of plate culture medium per well.c. Prepare LipoInsect™ Transfection Reagent/Bacmid complex as indicated in the table below. For each well to be transfected, dilute 16µg bacmid and 8µl of LipoInsect™ Transfection Reagent with 100µl of SFX-INSECT insect cell culture medium (without antibiotics and serum) in sterile tubes, respectively, and mix gently by pipetting or vortexing (Do not centrifuge). After incubation at room temperature for 2-5 minutes (no more than 30 minutes), mix the diluted bacmid with the diluted Transfection Reagent gently and incubate at room temperature for 15-30 minutes.96-well48-well24-well12-well6-well6cm dish10cm dishLipo6000™ Transfection Reagent0.32μl0.8μl1.6μl3.2μl8μl16μl48μlCulture medium (without antibiotics and serum)4μl10μl20μl40μl100μl200μl600μlBacmid640ng1.6μg3.2μg6.4μg16μg32μg96μgCulture medium (without antibiotics and serum)4μl10μl20μl40μl100μl200μl600μlTransfer the diluent bacmid into the diluted transfection reagent. Mix gently and incubate at room temperature for 15-30 minutes.Note 1: Bacmid amount can be appropriately adjusted from 8μg to 24μg per well in 6-well plates. The optimal bacmid amount varies depending on the cell type and culture conditions and can be optimized within the range recommended above.Note 2: The recommended concentration of bacmid should be in the range of 0.5-5μg/μl prior to dilution.Note 3: Prepare a master mix when transfecting multiple cell samples with the same bacmid at the same amount, and then dispense the recommended amount to each well.Note 4: For other culture plates or petri dishes, the dose of each reagent can be adjusted proportionally based on the culture area of the culture format (Appendix).a. Add 200µl of Transfection Reagent/Bacmid complexes drop-wise to each well of a 6-well plate. Gently shake the culture plate to evenly distribute the transfection complexes.b. In order to achieve the maximum transfection efficiency, please replace the transfection mixture with a fresh complete culture medium after 3-5 hours of transfection (For Sf9 cells, we recommend changing the culture medium after 4 hours ofr transfection).c. After 96 hours of incubation at 27-28℃, harvest cells by centrifuge at 500×g for 5 minutes. The obtained supernatant contains P1 baculovirus with a viral titer of 1×106-1×107pfu/ml. It should be stored at 4℃ in the dark, or stored at -80℃ for long-term storage. Repeated freeze-thaw will decrease the viral titer by 10-100 times, which should be avoided as possible.Note: Serum proteins act as substrates for proteases and prevent virus degradation. If the culture medium for packaging virus is serum-free, please add 2% serum. d. To increase the viral titer, it is recommended to transfect Sf9 or Sf21 cells with P1 baculovirus. Generally, the obtained P2 baculovirus has a viral titer of 1×107-1×108pfu/ml. The general procedure is to infect 2×106 Sf9 or Sf21 cells (which have adhered for about 1 hour) with P1 baculovirus with the recommended MOI of 0.05-0.1. A high MOI results in decreased quality of obtained virus. After incubation at 27-28ºC for 48 hours, collect the culture medium of each well, and centrifuge at 500×g for 5 minutes. The obtained supernatant contains P2 baculovirus. The storage conditions for P2 baculovirus are the same as for P1 baculovirus. Since each amplification of baculovirus may cause the loss and mutation of genes, up to three generations at most can be amplified. Note: For detailed procedures of amplifying baculovirus, please refer to the protocols of the Bac-to-Bac Baculovirus expression system for insect cells.FAQ: 1. Low transfection efficiency:a. Optimize the ratio of bacmid to LipoInsect™ Transfection Reagent. Increase the amount of bacmid for cells that are difficult to transfect.b. Use highly purified, sterile, and contaminant-free bacmid for transfection. High-purity bacmid should have an A260/A280 ratio ranging from 1.8 to 1.9. Preparing bacmids using the solution I, II, and III from the Plasmid Preparation Kit ( Transfection Reagent/Bacmid complexes should be prepared in a culture medium without antibiotics and serum.e. Insufficient incubation time after transfection may result in low transfection efficiency. The recommended incubation time for high transfection efficiency is 72-96 hours.f. Regularly check cells for mycoplasma infection. Mycoplasma contamination detrimentally affects cell proliferation, and thereby the transformation efficiency.g. If the expression of the target protein is not detected, check the sequence of the plasmid to ensure the open reading frame is correct.2. Cellular toxicity occurs:a. Decrease the transfection time and replace a fresh cell culture medium within a relatively short time after transfection.b. Reduce the amount of bacmid and also LipoInsect™ Transfection Reagent proportionally. c. Check if the cell density is too low at the time of transfection.Appendix:The size, culture area, cell culture volume, and recommended culture volume of commonly used multi-well plates and petri dishes:Multiple Well Plates or DishesSingle Well Only for PlatesDiameter(Bottom, mm)*Growth Area(cm2)*AverageCell YieldTotal WellVolume (ml)WorkingVolume (ml)Recommended Volume (ml)6 well34.89.59.5 × 10516.81.9-2.9212 well22.13.83.8 × 1056.90.76-1.14124 well15.61.91.9× 105 3.40.38-0.570.548 well11.00.959.5× 104 1.60.19-0.2850.2596 well6.40.323.2 × 1040.360.10-0.200.1384 well2.70.0565.6 × 1030.1120.025-0.0500.0301536 well1.63 × 1.63**0.0252.5 × 1030.1250.005-0.0100.0103.5 cm dish3499.0 × 105NA1.8-2.726 cm dish52212.1 × 106NA4.2-6.3510 cm dish8.4555.5 × 106NA11-16.51215cm dish141521.5 × 107NA30.4-45.63524.5cm dish22.4 × 22.4**5005.0 × 107NA100-150120*Diameter and growth area may vary depending on the manufacturer, and the listed sizes are from Corning. **These wells or dishes are square.

Specification: BioReagent, Suitable for molecular biology, sterile