Adult stage O. viverrini flukes from hamsters were washed several times with sterile normal saline and then washed five times in DMEM (Gibco, USA) containing 2× antibiotics. Flukes were incubated for 1 hour at 37°C in 5% CO2 atmosphere with DMEM containing 2× antibiotics. The flukes were resuspended in 100 μl of DMEM plus 1x antibiotic and 50 μg of Ov-grn-1 dsRNA in 4 mm gap cuvettes (Bio-Rad, Hercules, CA, USA) and subjected to a single pulse of square wave electroporation at 125 V for 20 milliseconds (Electroporator Gene Pulser Xcell, Bio-Rad). The parasites were soaked with 50 μg of Ov-grn-1 dsRNA in 10 ml of 1× DMEM for two days and maintained at 37°C and 5% CO2 in air before downstream processing.
4 mm gap cuvette
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The 4 mm gap cuvette is a laboratory equipment designed for spectrophotometric analysis. It provides a 4 mm path length for the sample, allowing for precise optical measurements.
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5 protocols using 4 mm gap cuvette
1
Electroporation-Mediated RNAi in O. viverrini Flukes
2
Transfection of Leishmania Promastigotes
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Clones were transfected by electroporation as described previously by Robinson and Beverley [36 (link)]. Briefly, 1 x 108 mid-log phase cells (per transfection) were pelleted at 1000g for 10 minutes and resuspended in half the original volume in ice cold Cytomix buffer (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4, 25 mM HEPES, 2 mM EDTA, 2 mM MgCl2; pH 7.6). Cells were pelleted again and resuspended in ice cold Cytomix buffer to a final concentration of 2 x 108 cells/ml. 8–10 μg of pLPneo2-HAT2 or pLPneo2 plasmids were aliquoted into 4 mm gap cuvettes (Bio-Rad) and 500 μl of cells were added to each cuvette and mixed by pipetting. Electroporation was done twice at 25 μF, 1500 V (3.75 kV/cm) with a gap of 10 seconds between pulses (Gene Pulser Xcell Electroporator, Bio-Rad). The electroporated cells were incubated on ice for 10 minutes and transferred in a flask with 10 ml M199 medium supplemented with 20% FBS. The flask was incubated at 25°C. After 24 hours, media was replaced with an additional supplementation of 100 μg/ml G418 (HiMedia) for selection of over-expressing cells.
3
TCRα/β Expression in Jurkat Cells
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TCRα/β expression vectors were transferred via electroporation into TCR deficient and CD8α expressing Jurkat cells (J76-CD8α)63 kindly provided by Dr. Wolfgang Uckert (Max Delbruck center, Berlin). J76-CD8α cells were maintained in RPMI1640 media containing 10% FBS in early log phase for transfection. Four million J76-CD8 cells (107/ml) were harvested and mixed with 10 – 15 µg of expression vector plasmid in a 4 mm gap cuvette (Biorad) for electroporation. Transfection was performed using BTX electroporator (260 V, 1050 µF). Thirty-six hours after transfection, cells were harvested for MHC-tetramer staining.
4
Overexpression of TCF21 in 786-O Cells
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The coding sequence of TCF21 (including HA‐tag) was cloned out of a pCS2+‐TCF21 construct [kindly provided by Prof. Christopher Plass and Khalifa Arab, Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany], amplified, and recloned into a pBABE‐puro vector. Plasmid DNAs were sequence‐confirmed. Twenty‐five micrograms of pBABE‐TCF21‐HA or pBABE‐puro vector alone was transfected into cells of the 786‐O cell line using electroporation. Electroporation was performed in a 4‐mm gap cuvette (#165‐2088; Bio‐Rad Laboratories, Hercules, CA, USA) using a Gene Pulser (Bio‐Rad, München, Germany) with electric parameters 24 kV with 1000 uF capacitance; a single exponential decay pulse was applied. Selection medium containing puromycin was added to the cells after 48 h of recovery, and colonies grew after 2 weeks of culture. Eight colonies were selected for functional assays: four from pBABE‐puro mock‐transfected 786‐O cells (N1F4, B5F1, N1G4, and B6D10) and four expressing HA‐tagged exogenous TCF21 (2B12, 5D2, 9D12, and 9H9). Unless the clones are specifically named, data from ‘pBABE‐mock’ or ‘pBABE‐TCF21’ contain pooled data from all four clones.
5
TCRα/β Expression in Jurkat Cells
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