Mice were injected with 0.5 μg–2.5 μg 9C12 in 100 μL endotoxin free PBS (Teknova) by lateral tail vein injection. 4h–24h later mice were injected with the indicated dose of Ad5 in 100 μL endotoxin free PBS (Teknova) by lateral tail vein injection. For macrophage depletion, mice were injected i.v. with 100μL clodronate liposomes (Liposoma) and infected with Ad5 48h after liposome injection.
Endotoxin free pbs
Manufactured by Teknova
Sourced in United States
Endotoxin-free PBS is a phosphate-buffered saline solution that has been purified to remove endotoxins. It is a commonly used buffer in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Pd 10 column, by GE Healthcare (3 mentions)
Ni nta, by Qiagen (2 mentions)
Noti hf restriction enzyme, by New England Biolabs (1 mentions)
Endofree plasmid purification kit, by Qiagen (1 mentions)
Clodronate liposome, by Liposoma (1 mentions)
Histrap hp 1 ml column, by GE Healthcare (1 mentions)
Electronic digital caliper, by Avantor (1 mentions)
6 protocols using endotoxin free pbs
1
Adenoviral Infection Model in Mice
2
Cloning and Expression of LdFeSODB1 Gene
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Leishmania donovani iron superoxide dismutase B1 (LdFeSODB1) gene was cloned into two different plasmid vectors. For DNA vaccine, the coding region of the gene was cloned into a modified pcDNA plasmid with or without mGMSCF fusion DNA. A spacer fragment was placed between mGMCSF and LdFeSODB1 genes. The spacer region possesses six histidine residues for recombinant protein purification, an enteropeptidase cleavage site, and flanking proline residues at both ends. After PCR amplification, the LdFeSODB1 gene and also the plasmid were digested with NotI-HF® restriction enzyme (New England BioLabs, Canada). After ligation of the PCR product and the plasmids, transformation of E. coli DH5α was performed. Confirmation of the cloning was done by sequencing of plasmid DNA extracted from transformed E. coli.
Endotoxin-free vaccine candidate plasmid DNA was isolated from the transformed E. coli DH5α using EndoFree® plasmid purification kit (QIAGEN, Canada) following the manufacturer's instruction. Endotoxin-free plasmid DNA samples were diluted to appropriate concentration using endotoxin-free PBS (Teknova, USA) and were used for protein expression study in Chinese Hamster Ovary cells (CHO) and for injection into mice. For expression in bacterial system, LdFeSODB1 was cloned into pET17b plasmid (Novagen) following previously published procedure [18 (link)].
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Leishmania donovani iron superoxide dismutase B1 (LdFeSODB1) gene was cloned into two different plasmid vectors. For DNA vaccine, the coding region of the gene was cloned into a modified pcDNA plasmid with or without mGMSCF fusion DNA. A spacer fragment was placed between mGMCSF and LdFeSODB1 genes. The spacer region possesses six histidine residues for recombinant protein purification, an enteropeptidase cleavage site, and flanking proline residues at both ends. After PCR amplification, the LdFeSODB1 gene and also the plasmid were digested with NotI-HF® restriction enzyme (New England BioLabs, Canada). After ligation of the PCR product and the plasmids, transformation of E. coli DH5α was performed. Confirmation of the cloning was done by sequencing of plasmid DNA extracted from transformed E. coli.
Endotoxin-free vaccine candidate plasmid DNA was isolated from the transformed E. coli DH5α using EndoFree® plasmid purification kit (QIAGEN, Canada) following the manufacturer's instruction. Endotoxin-free plasmid DNA samples were diluted to appropriate concentration using endotoxin-free PBS (Teknova, USA) and were used for protein expression study in Chinese Hamster Ovary cells (CHO) and for injection into mice. For expression in bacterial system, LdFeSODB1 was cloned into pET17b plasmid (Novagen) following previously published procedure [18 (link)].
3
Endotoxin-Free Protein Purification
4
Protective Immunogenicity of DNA and Recombinant Protein Vaccine Regimens against Leishmania major Infection in BALB/c Mice
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After two weeks of acclimatization, five female BALB/c mice per group were injected with DNA vaccine antigens or controls. Two DNA immunizations were given at week-0 and week-3 followed by one recombinant protein boost at week-6. All immunizations were prepared in 50 µl solution in endotoxin-free PBS (Teknova, USA). DNA immunization was performed by intramuscular injection of a mixture of 100 µg plasmid DNA and 25 µg CpG ODN in 50 µl total volume. The recombinant protein booster immunization was given to the vaccine groups by subcutaneous injection (SC) of 12.5 µg rLdPxn1 protein in combination with 25 µg CpG ODN in the right hind footpad. All the three injections to mice that received pcDNA and pcDNA-mGMCSF were given in the form of plasmid DNA only.
Leishmania major strain V1 (MHOM/IL/80/Friedlin) was used for the protection study in BALB/c mice. Stationary phase promastigotes, cultured in M199 medium with 20% FBS, were washed and resuspended in endotoxin-free PBS. 3×106 stationary phase live promastigotes in 40 µl endotoxin-free PBS were injected subcutaneously into the hind left footpad of each mouse. The thickness of the footpads was then measured weekly until euthanasia using an electronic digital caliper (VWR, USA). Mice that showed a net footpad swelling of more than 3 mm thick or those that developed necrotic lesions were euthanized even before the end date of week-17.
Leishmania major strain V1 (MHOM/IL/80/Friedlin) was used for the protection study in BALB/c mice. Stationary phase promastigotes, cultured in M199 medium with 20% FBS, were washed and resuspended in endotoxin-free PBS. 3×106 stationary phase live promastigotes in 40 µl endotoxin-free PBS were injected subcutaneously into the hind left footpad of each mouse. The thickness of the footpads was then measured weekly until euthanasia using an electronic digital caliper (VWR, USA). Mice that showed a net footpad swelling of more than 3 mm thick or those that developed necrotic lesions were euthanized even before the end date of week-17.
5
Purification of Recombinant VHHs from E. coli
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WK6 E. coli containing the plasmid encoding VHHs were grown to midlog phase at 37 °C in Terrific Broth plus ampicillin and induced with 1 mM isopropyl β-d -1-thiogalactopyranoside overnight at 30 °C. Bacteria were harvested by centrifugation at 5,000g for 15 min at 4 °C and then resuspended in 25 mL 1× TES buffer (200 mM Tris, pH 8, 0.65 mM EDTA, 0.5 M sucrose) per liter of culture and incubated for 1 h at 4 °C with agitation. Resuspended cells were then submitted to osmotic shock by 1:4 dilution in 0.25× TES buffer and incubation overnight at 4 °C. The periplasmic fraction was isolated by centrifugation at 5,000g for 30 min at 4 °C and then loaded onto Ni-NTA (Qiagen) in 50 mM Tris, pH 8, 150 mM NaCl, and 10 mM imidazole. Protein was eluted in 50 mM Tris, pH 8, 150 mM NaCl, 500 mM imidazole, and 10% glycerol and then loaded onto a Superdex 75 10/300 column in 50 mM Tris, pH 8, 150 mM NaCl, and 10% glycerol. The peak fractions were recovered and rebound to Ni-NTA for depletion of lipopolysaccharide (<2 IU/mg). Bound VHHs were washed with 40 column volumes of PBS + 0.1% TritonX-114 and eluted in 2.5 column volumes of endotoxin-free PBS (Teknova) with 500 mM imidazole. Imidazole was removed by size exclusion on a PD10 column (GE Healthcare) and elution in lipopolysaccharide-free PBS. Purity of recombinant VHHs was assessed by SDS-PAGE and LC-MS.
6
Recombinant VHH Purification and Endotoxin Removal
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Expression of VHHs and endotoxin removal WK6 E. coli containing the plasmid encoding corresponding VHHs were grown to mid-log phase at 37°C in Terri c Broth plus ampicillin and induced with 1 mM IPTG overnight at 30°C. Bacteria were harvested by centrifugation at 5,000g for 15 minutes at 4 °C and then resuspended in 25 mL 1× TES buffer (200 mM Tris, pH 8, 0.65 mM EDTA, 0.5 M sucrose) per liter culture and incubated for 1 hour at 4 °C with agitation. Resuspended cells were then submitted to osmotic shock by 1:4 dilution in 0.25× TES buffer and incubation overnight at 4 °C. The periplasmic fraction was isolated by centrifugation at 5,000g for 30 min at 4 °C and then loaded onto Ni-NTA (Qiagen) in 50 mM Tris, pH 8, 150 mM NaCl, and 10 mM imidazole. Protein was eluted in 50 mM Tris, pH 8, 150 mM NaCl, 500 mM imidazole, and 10% glycerol and then loaded onto a Superdex 75 10/ 300 column in 50 mM Tris, pH 8, 150 mM NaCl, 10% glycerol.
The peak fractions were recovered and rebounded to Ni-NTA to be depleted of LPS (<2 IU/mg). Bound VHHs were washed with 40 column volumes of PBS + 0.1% TritonX-114 and eluted in 2.5 column volumes endotoxin-free PBS (Teknova) with 500 mM imidazole. Imidazole was removed by PD10 column (GE Healthcare), eluting in LPS-free PBS. Recombinant VHH purity was assessed by SDS/PAGE and LC-MS.
The peak fractions were recovered and rebounded to Ni-NTA to be depleted of LPS (<2 IU/mg). Bound VHHs were washed with 40 column volumes of PBS + 0.1% TritonX-114 and eluted in 2.5 column volumes endotoxin-free PBS (Teknova) with 500 mM imidazole. Imidazole was removed by PD10 column (GE Healthcare), eluting in LPS-free PBS. Recombinant VHH purity was assessed by SDS/PAGE and LC-MS.
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