For IM-EP delivery, mice were injected intramuscularly with 50 μg of HER2 DNA vaccines (pVAX1-HER2) per mouse in a final volume of 50 μl of PBS using a 31-gauge needle. The injections were followed by EP at 0.2 volts for 4 sec using Cellectra® of VGX International Inc./Inovio in accordance with the manufacturer's protocol. The HER2 DNA vaccine, which coded for an extracellular region of the human HER2 protein, was kindly provided by W.Z. Wei (Wayne State University, Detroit, MI). Plasmid DNA was produced in bacteria and purified using endotoxin-free Qiagen kits according to the manufacturer's protocol (Qiagen, Valencia, CA).
Endotoxin free kit
Manufactured by Qiagen
Sourced in United States, Spain
The Endotoxin-free kit is a laboratory product designed to remove endotoxins from biological samples. It is a tool used in the purification process to ensure the sample is free from endotoxin contamination.
Automatically generated - may contain errors
Lab products found in correlation
Effectene transfection reagent, by Qiagen (2 mentions)
Pegfp n1, by Takara Bio (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Poly hema, by Merck Group (1 mentions)
Poly 1 c, by Merck Group (1 mentions)
Helios gene gun system, by Bio-Rad (1 mentions)
19 protocols using endotoxin free kit
1
Intramuscular HER2 DNA Vaccine Delivery
2
DNA Vaccine Delivery via Intramuscular Electroporation
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For intramuscular (IM)-electroporation (EP) delivery of DNA vaccines, mice were injected intramuscularly with 50 µg of pcDNA3-GP per mouse in a final volume of 50 µL of phosphate-buffered saline (PBS) using a 31-gauge needle. The injections were followed by EP at 0.2 volts for 4 seconds using Cellectra of GeneOne Life Science (Seoul, Korea) in accordance with the manufacturer's protocol. Plasmid DNA was produced in bacteria and purified using endotoxin-free Qiagen kits according to the manufacturer's protocol (Qiagen, Valencia, CA, USA). For delivery of recombinant GP, mice were injected intraperitoneally with 20 µg of GP per mouse emulsified in 400 µL of incomplete Freund's adjuvant. Mice were also injected intravenously with 20 µg of recombinant GP per mouse in a final volume of 200 µL of PBS.
3
Neurosphere Culture and Characterization
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Neurosphere cultures were derived from E13-E14 embryonic and adult mouse spinal cord as previously described in [11 (link),12 (link)] and grown in defined media supplemented with EGF/FGF2. Cell growth was measured by seeding dissociated cells (1000 or 5000 cells per well) in 1 ml of media in 24-well plates coated with poly-HEMA (Sigma) to inhibit cell adherence. After 5 or 7 days, the neurospheres were directly dissociated by addition of trypsin in the wells (0.5% final) and the cell number was measured with an automated cell counter (Z2, Beckman Coulter). To determine the neurosphere forming unit (Nsfu), the neurospheres were enzymatically dissociated and clonally seeded at 1 cell/well in 96-well plates using an automatic cell seeding device (Aria cytometer BD). After two weeks, the number of spheres with a diameter >500μm was visually determined. Nsfu = number of spheres/96 wells x 100. Neurosphere differentiation was obtained by culturing them for 4 days without growth factor on poly-D-lysine coated coverslips. Neurosphere transfection was performed using an Amaxa apparatus with a neural stem cell tranfection kit (Amaxa). ErbB2 (Addgene n°10917) and control GFP plasmids were obtained from Addgene and amplified using endotoxin-free kits (Qiagen). Purified recombinant OCAM coupled to Fc fragment (OCAM-Fc) and control Fc fragment, produced in CHO cells, were purchased from R&D.
4
Influenza Virus Matrix Protein-Based RSV VLP
5
Production and Characterization of RSV VLP
6
Construction of Cdc42 Expression Plasmids
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The reporter plasmid pCAGGs: EGFP was a kind gift from Elly M. Tanaka11
. To construct pCAGGs: Cdc42 plasmid, Cdc42 coding sequence was PCR-amplified from axolotl brain cDNA with primer pair Cdc42-Fw (5′-ATGCAGACAATTAAATGTGTAGTTGTTGGG-3′) and Cdc42-Rev (5′-TCATAGCAGCACACACTTGCG-3′). Cdc42 PCR product was subcloned into pCAGGs vector. To construct pCAGGs: null plasmid for control, Cdc42 coding sequence was deleted from plasmid pCAGGs: Cdc42. All plasmids were verified by sequencing and purified with Endotoxin-free kit (12162, Qiagen, Hilden, Germany) following the manufacturer’s instructions.
. To construct pCAGGs: Cdc42 plasmid, Cdc42 coding sequence was PCR-amplified from axolotl brain cDNA with primer pair Cdc42-Fw (5′-ATGCAGACAATTAAATGTGTAGTTGTTGGG-3′) and Cdc42-Rev (5′-TCATAGCAGCACACACTTGCG-3′). Cdc42 PCR product was subcloned into pCAGGs vector. To construct pCAGGs: null plasmid for control, Cdc42 coding sequence was deleted from plasmid pCAGGs: Cdc42. All plasmids were verified by sequencing and purified with Endotoxin-free kit (12162, Qiagen, Hilden, Germany) following the manufacturer’s instructions.
7
Heterologous DNA/Vaccinia Boost Immunization
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The DNA vaccines pcDNA3-CRT/16E6, pcDNA3-CRT/16E6(R55K), and pcDNA3-CRT/16E6(R55K)(delK75) were prepared by using an endotoxin-free kit (Qiagen, Valencia, CA). TA-HPV is a recombinant vaccinia virus expressing HPV16/18 E6/E7, and it was described previously (40 (link)). DNA was given to C57BL/6 mice via intramuscular (i.m.) injection followed by EP using an Electro Square Porator (Holliston, MA) at the hind leg muscle. The mice were boosted once 7 days later with the same regimen. Seven days after the second DNA vaccination, the mice were further boosted with TA-HPV via skin scarification, as described previously (41 (link)).
8
Generation of Pkt2-Luc-T2a-HPV18E7E6(del D70)
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The generation of pKT2/CLP-AKT plasmid [32 (link)] and pCMV(CAT)T7-SB100 plasmid [33 (link)] has been described previously. These plasmids were purchased from Addgene. The generation of Pkt2-cMyc has been described previously [31 ]. To generate Pkt2-Luc-T2a-HPV18E7E6(del D70), 18E6 (delD70) was first amplified via PCR using the Pkt2-LucHPV18E7E6 [34 (link)] template and the following set of primers: 5′-CTGGCTCGAGGAGGGAAGGGGAAGCCTGCT-3′, 5′-GCTCCCGGATTCTGCTGTAGAAGATACACTTGTGGCAAGCGGCG-3′, 5′-CGCCGCTTGCCACAAGTGTATCTTCTACAGCAGAATCCGGGAGC-3′, AND 5′-AAACCAGCTAGCTGGTTATTACACCTGGGTCTC-3′. The amplified PCR product was then cloned into the Xho/bstX1 sites of a Pkt2-LucHPV18E7E6. Plasmid construct was confirmed using DNA sequencing and the DNA was prepared using an endotoxin-free kit (QIAGEN, Valencia, CA, USA).
9
Transient transfection of CHIP mutants
10
Indoleamine 2,3-dioxygenase Activity Assay
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Bacterial pDNA (pEGFPN1, Clontech) was prepared using an endotoxin-free Kit (Qiagen). Mice were intravenously injected with 30 μg pDNA mixed with in vivo-jetPEI (Polyplus -transfection, N:P =8) or were injected s.c. with NPs (150 μg, 5-fold of vaccine dose). IDO activity was measured as described in previous reports40 (link),41 (link). Enzyme activity was expressed as the product content per hour per gram of tissue protein.
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