Filtered T. gondii were washed and resuspended in Cytomix (10 mM KPO4, 120 mM KCl, 5 mM MgCl2, 25 mM HEPES, 2 mM EDTA) at 2×108 parasites/ml. 5×107 parasites were combined with 100 µg of CRISPR/Cas9 plasmid supplemented with 2 mM ATP, 5 mM GSH, and 150 µM CaCl2, with or without 40 µg of double-stranded oligonucleotide, in a final volume of 400 µl. The CaCl2 concentration used here matches the original formulation of Cytomix [8] (link) but was added at the time of transfection to prevent the buildup of phosphate precipitates during buffer storage. Parasites were electroporated in 4 mm gap cuvettes (BTX Harvard Apparatus model no. 640) in an Electro Square Porator (BTX Harvard Apparatus). 200 parasites were added to confluent HFF monolayers in six-well dishes. In some cases, as indicated in the text, 2,000 or 20,000 parasites were used instead. If indicated, 0.2 µM Compound 2 (C2) or vehicle (DMSO) was added to these dishes 24 hpi. All plates were allowed to incubate for a total of eight days before staining with crystal violet. C2 was synthesized by N. Westwood and F. Tran as previously described [4] (link), [9] (link), [10] (link).
Electro square porator
Manufactured by Harvard Apparatus
Sourced in United States
The Electro Square Porator is a lab equipment device designed for electroporation, a technique used to introduce foreign molecules into cells. The device generates square-wave electrical pulses that create temporary pores in cell membranes, facilitating the uptake of materials such as DNA, RNA, or proteins. The Electro Square Porator's core function is to provide a controlled electrical input for this cell manipulation process.
Automatically generated - may contain errors
Lab products found in correlation
Fast green, by Merck Group (4 mentions)
Polycarbonate filter, by Cytiva (2 mentions)
Cytomix, by Cytiva (2 mentions)
Model no 640, by Harvard Apparatus (2 mentions)
Mm tweezertrodes, by Harvard Apparatus (2 mentions)
4 0 vicryl, by Johnson & Johnson (1 mentions)
Cuy650p3, by Nepa Gene (1 mentions)
Pulled glass capillaries, by Harvard Apparatus (1 mentions)
9 protocols using electro square porator
1
Efficient Genetic Modification of T. gondii Using CRISPR-Cas9
2
In Vivo Electroporation of Chick Embryos
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Chick embyros were electroporated at E2.5 using fine platinum electrodes and an ElectroSquare Porator, Harvard Apparatus, Massachusetts, USA. Settings were as follows; 15 V, 3 pulses, 50 ms pulse duration, 950 ms interval. DNA was diluted to a final concentration of 1 μg/ml and fast green added to aid visualization. DNA injected into the neural tube at diencephalon level; electrodes were placed either side of the diencephalon over the thalamus or prethalamus. DNA overexpression vectors plasmids used; pCAGGS-Helt-ires-eGFP, pCAGGS-Sox14-ires-eGFP, pCAGGS-Dlx2 (gift from John L Rubenstein, UCSF) (co-electroporated with pCAGGS-eGFP), pRFPSox14i(1-4), pRFPscrambled and p3XFLAGSox14.
3
Inducing Muscle Degeneration: Electroporation and Cardiotoxin
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To induce muscle degeneration, we used two different techniques. The first one is based on the methodology for electroporation of the muscle, using an electrical shock-induced injury protocol (24) .
Briefly, the hairs from the calves were removed and the electrodes pads were positioned perpendicularly to muscle fibers orientation, in the center of the muscles, at equal distances from the knee and the ankle.
The muscles were injured by applying eight electrical pulses at an intensity of 100 Volts, during 20 milliseconds each pulse, separated by a 0.5-second interval delivered using an electroporation device (ECM830, Electro Square Porator, Harvard Apparatus, Holliston, MA, USA). The second injury procedure was done by cardiotoxin injection (25) . We injected 150 μL of 10 μM cardiotoxin solution percutaneously (Cardiotoxin, Naja pallida, 217503, Merck, Molsheim, Alsace, France) in both gastrocnemius muscles, in the center of the muscles, at equal distances from the knee and the ankle. Animals were euthanized by cervical dislocation three, five, ten, and fifteen days post-lesion. For each mouse, one muscle was used for histological analysis, and the contralateral muscle was used for molecular analysis.
Briefly, the hairs from the calves were removed and the electrodes pads were positioned perpendicularly to muscle fibers orientation, in the center of the muscles, at equal distances from the knee and the ankle.
The muscles were injured by applying eight electrical pulses at an intensity of 100 Volts, during 20 milliseconds each pulse, separated by a 0.5-second interval delivered using an electroporation device (ECM830, Electro Square Porator, Harvard Apparatus, Holliston, MA, USA). The second injury procedure was done by cardiotoxin injection (25) . We injected 150 μL of 10 μM cardiotoxin solution percutaneously (Cardiotoxin, Naja pallida, 217503, Merck, Molsheim, Alsace, France) in both gastrocnemius muscles, in the center of the muscles, at equal distances from the knee and the ankle. Animals were euthanized by cervical dislocation three, five, ten, and fifteen days post-lesion. For each mouse, one muscle was used for histological analysis, and the contralateral muscle was used for molecular analysis.
4
Electroporation of Freshly Egressed Parasites
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Freshly egressed parasites were passed through a polycarbonate filter with a 3-μm pore size (Whatman), then washed, and resuspended in Cytomix (34 (link)) (10 mM KPO4, 120 mM KCl, 150 mM CaCl2, 5 mM MgCl2, 25 mM HEPES, 2 mM EDTA) to 8 × 107 cells/ml. Two hundred forty-five microliters of the parasite suspension was combined with 50 μg of previously linearized plasmid in 155 μl Cytomix, supplemented with 2 mM ATP and 5 mM glutathione (GSH), to a final volume of 400 μl. Parasites were electroporated in 4-mm-gap cuvettes (BTX Harvard Apparatus model no. 640) in an Electro Square Porator (BTX Harvard Apparatus) set to 1.7 kV, for two 176-μs pulses at 100-ms intervals.
5
Electroporation of Plasmodium Parasites
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Freshly egressed parasites were passed through a polycarbonate filter with a 3-μm pore size (Whatman) and then washed and resuspended in Cytomix (38 (link)) (10 mM KPO4, 120 mM KCl, 150 mM CaCl2, 5 mM MgCl2, 25 mM HEPES, 2 mM EDTA) to 8 × 107 cells/ml. Two hundred forty-five microliters of the parasite suspension was combined with 50 μg of previously linearized plasmid in 155 μl Cytomix, supplemented with 2 mM ATP, 5 mM glutathione (GSH), to a final volume of 400 μl. Parasites were electroporated in 4-mm-gap cuvettes (BTX Harvard Apparatus model no. 640) in an Electro Square Porator (BTX Harvard Apparatus) set to 1.7 kV, for two 176-μs pulses at 100-ms intervals.
6
In Utero Neonatal Brain Electroporation
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Electroporations were performed on pregnant CD1 (ICR) mice (E15, Charles River ca. SC:022). For each surgery, the mouse was initially anesthetized with 5% isoflurane and maintained with 2.5% isoflurane. The surgery was conducted on a heating pad, and warm sterile phosphate-buffered saline (PBS) was intermittently perfused over the pups throughout the procedure. A micropipette was used to inject ~2 μl of recombinant DNA at a concentration of 2 μg/μl and into the left ventricle of each neonate’s brain (typically DNA encoding opsins were doped with plasmids expressing GFP or mRuby3 at a concentration of 1:20 to facilitate screening for expression). Fast-green (Sigma-Aldrich) was used to visualize a successful injection. Following successful injection, platinum-plated 5mm Tweezertrodes (BTX Harvard Apparatus ca. 45–0489) were positioned along the frontal axis across the head of the neonate with the positive electrode of the tweezers positioned against the left side of the head. An Electro Square Porator (BTX Harvard Apparatus ca. 45-0052) was used to administer a train of 5 × 40 mV pulses with a 1s delay. After the procedure, the mouse was allowed to recover and come to term, and the delivered pups were allowed to develop normally.
7
In Utero Neonatal Brain Electroporation
8
In-utero Electroporation for Embryonic Gene Delivery
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In-utero electroporation was performed as reported previously (García-Moreno et al. 2014 (link)) with modifications. Time-mated pregnant dames were administered Vetergesic intraperitoneally (0.08 mg/kg) for analgesia prior to anesthesia with Isoflurane. A midline laparotomy was undertaken and uterine horns exposed gently. DNA (mixed with Fast Green, Sigma) was injected into the lateral ventricles using pulled glass capillaries (Harvard Apparatus). Square pulses (at 30–35 V, 5 × 50 ms with 950 ms resting periods) were delivered using tweezer electrodes (CUY650P3, Nepagene) connected to a square pulse generator (Electro Square Porator, Harvard Apparatus). Warm PBS was used to keep the embryo sacs at near body temperature along with a warm blanket.
Following electroporation, the uterine horns were gently placed back in the abdominal cavity. Surgical incisions were sutured (Vicryl 4/0, Ethicon) and the skin held together using surgical clips. Animals were allowed to recover after removal of anesthesia and kept on heating pads until they regained movement.
Following electroporation, the uterine horns were gently placed back in the abdominal cavity. Surgical incisions were sutured (Vicryl 4/0, Ethicon) and the skin held together using surgical clips. Animals were allowed to recover after removal of anesthesia and kept on heating pads until they regained movement.
9
In Utero Electroporation of Neonatal Mice
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Electroporations were performed on pregnant CD1 (ICR) mice (E15, Charles River ca. SC:022). For each surgery, the mouse was initially anesthetized with 5% isoflurane and maintained with 2.5% isoflurane. The surgery was conducted on a heating pad, and warm sterile PBS was intermittently perfused over the pups throughout the procedure. A micropipette was used to inject ~2 µl of recombinant DNA at a concentration of 2 µg/µl and into the left ventricle of each neonate’s brain (typically DNA encoding opsins were doped with plasmids expressing GFP or mRuby3 at a concentration of 1:20 to facilitate screening for expression). Fast-green (Sigma-Aldrich) was used to visualize a successful injection. Following successful injection, platinum-plated 5 mm Tweezertrodes (BTX Harvard Apparatus ca. 45-0489) were positioned along the frontal axis across the head of the neonate with the positive electrode of the tweezers positioned against the left side of the head. An Electro Square Porator (BTX Harvard Apparatus ca. 45-0052) was used to administer a train of 5 × 40 mV pulses with a 1 s delay. After the procedure, the mouse was allowed to recover and come to term, and the delivered pups were allowed to develop normally.
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