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Btx transfection device

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The BTX transfection device is a laboratory instrument designed for the introduction of foreign genetic material, such as DNA or RNA, into cells. The core function of the device is to facilitate the process of transfection, which is the delivery of nucleic acids into cells. The BTX transfection device provides the necessary electrical pulses or chemical reagents to enable the uptake of the genetic material by the target cells.

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Lab products found in correlation

Amaxa nucleofector, by Lonza (5 mentions) Quikchange 2 kit, by Agilent Technologies (1 mentions)

Spelling variants of this product

BTX device (2 citations)

5 protocols using btx transfection device

1

Cultivation and Transfection of Trypanosome Cell Lines

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BF cells were grown in HMI-9 (Hirumi and Hirumi 1989 (link)) with 10% FBS at 37°C, 5% CO2. PF cells were grown in SDM-79 (Brun and Schönenberger 1979 (link)) with 10% FBS at 27°C. For growth curve analysis, cell density was measured using a Coulter Counter. BF were reseeded at 2 × 105 cells/mL in 10 mL every day, while PF were reseeded at 2 × 106 cells/mL in 10 mL every 2 d. Transfections of BF cell lines with the Amaxa Nucleofector (Lonza), and of PF cell lines with the BTX transfection device (Harvard Apparatus, Inc.), were carried out as previously described (Merritt and Stuart 2013 (link)). Concentrations of drugs used for selection and tet-regulated expression of transgenes are as follows. For BF: 2.5 µg/mL G418, 5 µg/mL hygromycin, 2.5 µg/mL phleomycin, 0.5 µg/mL tet, 0.1 µg/mL puromycin. For PF: 15 µg/mL G418, 25 µg/mL hygromycin, 2.5 µg/mL phleomycin, 0.5 µg/mL tet, 1 µg/mL puromycin, 10 µg/mL blasticidin, 25 µg/mL ganciclovir.
McDermott S.M, & Stuart K. (2017). The essential functions of KREPB4 are developmentally distinct and required for endonuclease association with editosomes. RNA, 23(11), 1672-1684.
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2

Cultivation and Transfection of Trypanosoma Cell Lines

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BF cells were grown in HMI-9 (Hirumi and Hirumi 1989 (link)) with 10% FBS at 37°C, 5% CO2. PF cells were grown in SDM-79 (Brun and Schönenberger 1979 (link)) with 10% FBS at 27°C. For growth curve analysis, cell density was measured using a Coulter Counter. BF were reseeded at 2 × 105 cells/mL in 10 mL every day, while PF were reseeded at 2 × 106 cells/mL in 10 mL every 2 d. Transfections of BF cell lines with the Amaxa Nucleofector (Lonza), and of PF cell lines with the BTX transfection device (Harvard Apparatus, Inc.), were carried out as previously described (Merritt and Stuart 2013 (link)). Concentrations of drugs used for selection and tet-regulated expression of transgenes are as follows. For BF: 2.5 µg/mL G418, 5 µg/mL hygromycin, 2.5 µg/mL phleomycin, 0.5 µg/mL tet, 0.1 µg/mL puromycin. For PF: 15 µg/mL G418, 25 µg/mL hygromycin, 2.5 µg/mL phleomycin, 0.5 µg/mL tet, 1 µg/mL puromycin, 10 µg/mL blasticidin, 25 µg/mL ganciclovir, 100 µg/mL nourseothricin.
McDermott S.M., Carnes J, & Stuart K. (2019). Editosome RNase III domain interactions are essential for editing and differ between life cycle stages in Trypanosoma brucei. RNA, 25(9), 1150-1163.
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3

Cultivation and Transfection of Trypanosome Cell Lines

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BF cells were grown in HMI-9 (Hirumi and Hirumi, 1989 (link)) with 10% FBS at 37°C, 5% CO2. PF cells were grown in SDM-79 (Brun and Schonenberger, 1979 (link)) with 10% FBS at 27°C. Transfections of BF cell lines with the Amaxa Nucleofector (Lonza), and of PF cell lines with the BTX transfection device (Harvard Apparatus, Inc.), were carried out as described (Merritt and Stuart, 2013 (link)), with the exception that Tb-BSF buffer (90 mM sodium phosphate buffer (Na2HPO4/NaH2PO4), 5 mM KCl, 0.15 mM CaCl2, 50 mM HEPES pH 7.2) was used for BF nucleofection instead of the Human T Cell Nucleofector Kit (Schumann Burkard et al., 2011 (link)). Unless otherwise stated, concentrations of drugs used for selection and tetracycline (tet)-regulated expression of transgenes are as follows. For BF: 2.5 µg/mL G418, 5 µg/mL hygromycin, 2.5 µg/mL phleomycin, 0.5 µg/mL tet, 0.1 µg/mL puromycin, 5 µg/mL blasticidin, 12 µg/mL nourseothricin, 29 µg/mL trimethoprim. For PF: 15 µg/mL G418, 25 µg/mL hygromycin, 2.5 µg/mL phleomycin, 0.5 µg/mL tet, 1 µg/mL puromycin, 10 µg/mL blasticidin.
McDermott S.M., Pham V., Oliver B., Carnes J., Sather D.N, & Stuart K.D. (2024). Deep mutational scanning of the RNase III-like domain in Trypanosoma brucei RNA editing protein KREPB4. Frontiers in Cellular and Infection Microbiology, 14, 1381155.
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4

Site-Directed Mutagenesis for B4-3×V5 Expression

Cited 1 time
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The Gateway expression clone pHD1344tub(PAC)-KREPB4-Cterm3V5 (McDermott and Stuart, 2017 (link)) was used as a template for site-directed mutagenesis (QuikChange II kit; Agilent) using the primers listed in Supplementary Table 1. NotI-digested plasmids were transfected into the relevant BF and PF B4 CN cells (McDermott and Stuart, 2017 (link)). Transfections of BF cell lines with the Amaxa Nucleofector (Lonza) and of PF cell lines with the BTX transfection device (Harvard Apparatus, Inc.) were carried out as described above. Cell lines resistant to puromycin were selected, and constitutive expression of B4-3×V5 was confirmed by Western blotting. For standard growth curve analyses of independent cell lines, cell density + and - tet were measured using a Coulter Counter. BF were reseeded at 0.75 x 105 cells/mL in 10 mL every day, whilst PF were reseeded at 1 x 106 cells/mL in 10 mL every two days. To generate cell growth heat maps, cumulative growth numbers for - tet and + tet cultures were calculated for each time point. Next, the log2 of the ratio of - tet to +tet was calculated, and this number was converted to blue to orange scale using conditional formatting in Microsoft Excel.
McDermott S.M., Pham V., Oliver B., Carnes J., Sather D.N, & Stuart K.D. (2024). Deep mutational scanning of the RNase III-like domain in Trypanosoma brucei RNA editing protein KREPB4. Frontiers in Cellular and Infection Microbiology, 14, 1381155.
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5

Cell Culture Conditions for Bloodstream and Procyclic Trypanosomes

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BF cells were grown in HMI-9 (23 (link)) with 10% fetal bovine serum (FBS) at 37°C, 5% CO2. PF cells were grown at 27°C in SDM-79 (24 (link)) with 10% FBS, except for growth curve analyses, for which they were grown in MEM-Pros (25 (link)) with 10% dialyzed FBS plus or minus 6 mM d-glucose (26 (link)). For growth curve analyses, BF were reseeded at 2 × 105 cells/ml in 5 ml every day and PF were reseeded at 2 × 106 cells/ml in 10 ml every 2 days, and cell densities were measured using a Coulter Counter. Transfections of BF cell lines with the Amaxa Nucleofector (Lonza) and of PF cell lines with the BTX transfection device (Harvard Apparatus, Inc.) were carried out as described previously (27 (link)). Concentrations of drugs used for selection and tet-regulated expression of transgenes were as follows: BF were selected in 1 to 1.5 µg/ml hygromycin, 0.1 µg/ml puromycin, 5 µg/ml ganciclovir; PF were selected in 15 µg/ml G418, 25 µg/ml hygromycin, 2.5 µg/ml phleomycin, 0.5 µg/ml tet, 1 µg/ml puromycin, 10 µg/ml blasticidin, 25 µg/ml ganciclovir.
Carnes J., McDermott S.M, & Stuart K. (2018). RNase III Domain of KREPB9 and KREPB10 Association with Editosomes in Trypanosoma brucei. mSphere, 3(1), e00585-17.
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