DNA plasmids encoding HLA‐A*03:01 α‐chain and β2‐microglobulin were transformed separately into a BL21 strain of Escherichia Coli, as previously described.63 Recombinant proteins were expressed individually, where inclusion bodies were extracted and purified from the transformed E. coli cells. Soluble pHLA complexes were produced by refolding 30 mg of HLA‐A*03:01 with 10 mg of β‐2‐microglobulin and 5 mg of either NP265‐IAV, NP323‐IBV or NP270‐ICV peptide (Genscript, Piscataway, USA) into a buffer of 3 m Urea (Univar solutions, USA), 0.5 m L‐Arginine (Sigma‐Aldrich), 0.1 m Tris–HCl pH 8.0 (Fisher Bioreagents), 2.5 mm EDTA pH 8.0 (Sigma‐Aldrich, St Louis, USA), 5 mm Glutathione (reduced) (Goldbio, St Louis, USA) and 1.25 mm Glutathione (oxidised; Goldbio) for 3 h. The refold mixture was dialysed into 10 mm Tris–HCl pH 8.0 (Fisher Bioreagents), and soluble pHLA was purified using anion exchange chromatography using a HiTrapQ column (GE Healthcare).
Edta ph 8
Manufactured by Merck Group
Sourced in United States
EDTA pH 8.0 is a laboratory reagent used to chelate metal ions. It is a buffered solution of ethylenediaminetetraacetic acid (EDTA) at pH 8.0.
Automatically generated - may contain errors
Lab products found in correlation
Tris hcl ph 8, by Thermo Fisher Scientific (2 mentions)
Glutathione (gsh), by GoldBio (2 mentions)
Glutathione reduced, by GoldBio (2 mentions)
L arginine, by Merck Group (2 mentions)
Hitrap q column, by GE Healthcare (1 mentions)
Ecl prime reagent, by GE Healthcare (1 mentions)
Sodium chloride (nacl), by Carl Roth (1 mentions)
Anti hsp90 f 8, by Santa Cruz Biotechnology (1 mentions)
Anti foxo1 c29h4, by Cell Signaling Technology (1 mentions)
Na pyrophosphate, by Merck Group (1 mentions)
Mgcl2, by Carl Roth (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Nucleoside triphosphates, by Merck Group (1 mentions)
Ammonium sulfate, by Merck Group (1 mentions)
6 protocols using edta ph 8
1
Recombinant pHLA Complex Production
2
Splenic B Cell Protein Extraction and Western Blot
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2x106 magnetically-isolated splenic B cells were lysed in whole cell extraction buffer: 25 mM HEPES (Sigma-Aldrich), 0.3 M NaCl (Carl Roth), 1.5 mM MgCl2 (Carl Roth), 0.2 mM EDTA pH 8.0 (Sigma-Aldrich), 0.5% Triton X-100 (Sigma-Aldrich), 10 mM NaF (Sigma-Aldrich), 10 mM Na-pyrophosphate (Sigma-Aldrich), 100 μM Na-o-vanadate (Sigma-Aldrich), 2 mM DTT (Sigma-Aldrich), supplemented with protease inhibitors (cOmplete Protease Inhibitor Cocktail, Roche). 30-50 μg of proteins were separated by SDS-PAGE (31 (link)) and transferred onto PVDF membranes (Merck). Membranes were subsequently probed with anti-FOXO1 (C29H4, Cell Signaling Technology) and anti-HSP90 (F-8, Santa Cruz Biotechnology) primary antibodies, goat anti-mouse as well as anti-rabbit IgG secondary antibodies conjugated to HRP (Bio-Rad), and signals visualized using ECL prime reagent (GE Healthcare). Data were recorded on Chemidoc Imaging System (Bio-Rad) and analyzed using the Image Lab software (Bio-Rad).
3
Recombinant Peptide-HLA-B*15:01 Complex Production
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pET-30a(+) DNA plasmids encoding HLA-B*15:01 α-chain and human β2-microglobulin were transformed separately into BL21 E. coli and expressed. Inclusion bodies containing the individually expressed recombinant proteins were extracted, purified, and quantified as previously described.35 (link) Soluble peptide-HLA-B*15:01 complexes were produced by refolding 30 mg of HLA-B*15:01 α-chain with 10 mg of β2-microglobulin and 5 mg of peptide (Genscript, Piscataway, USA) into a buffer of 0.5 M L-Arginine (Sigma-Aldrich, St Louis, USA), 0.1 M Tris-HCl pH 8.0 (Fisher Bioreagents, Waltham, USA), 2.5 mM EDTA pH 8.0 (Sigma-Aldrich, St Louis, USA), 5 mM Glutathione (reduced) (Goldbio, St Louis, USA), 1.25 mM Glutathione (Goldbio, St Louis, USA). The peptides selected are summarized in Table 1 . The refold mixture was dialyzed at 4°C in 10 mM Tris-HCl pH 8.0 (3 times for 12 hours) and soluble pHLA complexes were purified via anion exchange chromatography.
4
Body Weight and Testosterone Analysis
5
Genotyping Mice by PCR and Enzyme Digestion
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Mice were genotyped by polymerase chain reaction (PCR) on tail biopsy samples using the following primers: forward, 5′-AGGCAGCGCCTTTGCTGCGTC-3′; reverse, 5′-TCCTGGTCACAGAGGTCCTTA-3′. PCR mix contained 1.0 μl of DNA, 0.2 μl of each primer, 0.4 μl of DreamTaq DNA Polymerase (Thermo Fisher Scientific, EP0703), and PCR buffer [containing tris-HCl (pH 8.8), ammonium sulfate (Sigma-Aldrich), MgCl2 (Sigma-Aldrich), 2-mercaptoethanol (Merck), EDTA (pH 8.0) (Sigma-Aldrich), nucleoside triphosphates (2′-deoxyadenosine 5′-triphosphate, 2′-deoxycytidine 5′-triphosphate, 2′-deoxyguanosine 5′-triphosphate, and 3′-deoxythymidine 5′-triphosphate) (Promega), bovine serum albumin (BSA; Ambion—Life Technologies), and H2O to a final volume of 20 μl]. PCR conditions were as follows: 3 min of initial denaturation at 94°C, followed by 36 cycles of 30-s denaturation at 94°C, 30-s annealing at 60°C, and 60-s elongation at 72°C. Final elongation was performed for 7 min at 72°C. Enzymatic digestion was performed with StyI (10 U/μl) (New England Biolabs, R0500S). The PCR product was separated by 3% gel electrophoresis (+/+ 600 bp, +/− 600 bp + 350 bp + 250 bp, −/− 350 bp + 250 bp).
6
Multiplex PCR for ESBL, EMA, and PMQR Detection
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PCR was performed using primers compatible with ESBL, EMA, and PMQR in all K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, and E. coli strains resistant to any antimicrobial drug tested in the AST following the work developed by Arlet, et al. (26) , Pitout, et al. (27) (link), van de Klundert, et al. (28) , and Jiang, et al. (29) (link).
Isolates showing resistance to carbapenems were sent to the Healthcare Associated Infections Laboratory (LAPIH) of the Oswaldo Cruz Institute (Fiocruz) for the identification of the bla KPC gene according to Yigit, et al. (30) (link).
The thermal cycling conditions were performed in a Cetus model 480 thermal cycler (Perkin-Elmer, Norwalk, CT).
PCR assays were performed in a 25 µl total volume adding the following components to the reaction tubes: 1 µl of target DNA (obtained from dilutions of colonies in 50 µl of 10 mM Tris, 1 mM EDTA -pH 8.0 (Sigma), 1.5 mM MgCl 2 (Invitrogen), 0,2 mM of dNTP mixture (dATP, dTTP, dCTP and dGTP) (Invitrogen), 20 pmol of each primer (Invitrogen), 1x PCR buffer (Promega), and 1.25 U of Taq DNA Polymerase (Invitrogen).
The amplified products were subjected to electrophoresis in a 1.5% agarose gel (Sigma). Table 1 shows all primers used in the PCR reactions and the expected amplicon sizes.
Isolates showing resistance to carbapenems were sent to the Healthcare Associated Infections Laboratory (LAPIH) of the Oswaldo Cruz Institute (Fiocruz) for the identification of the bla KPC gene according to Yigit, et al. (30) (link).
The thermal cycling conditions were performed in a Cetus model 480 thermal cycler (Perkin-Elmer, Norwalk, CT).
PCR assays were performed in a 25 µl total volume adding the following components to the reaction tubes: 1 µl of target DNA (obtained from dilutions of colonies in 50 µl of 10 mM Tris, 1 mM EDTA -pH 8.0 (Sigma), 1.5 mM MgCl 2 (Invitrogen), 0,2 mM of dNTP mixture (dATP, dTTP, dCTP and dGTP) (Invitrogen), 20 pmol of each primer (Invitrogen), 1x PCR buffer (Promega), and 1.25 U of Taq DNA Polymerase (Invitrogen).
The amplified products were subjected to electrophoresis in a 1.5% agarose gel (Sigma). Table 1 shows all primers used in the PCR reactions and the expected amplicon sizes.
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