Ultrafree cl centrifugal filter

The VH and VL genes of 9F4 were cloned into pFUSEss-CHIg-mG2a and pFUSE2ss-CLIg-mK cloning vectors (InvivoGen) respectively in order to generate mouse IgG2a wild type 9F4. Amino acid substitution K322A in the fragment crystallisable region (Fc region) of 9F4-pFUSEss-CHIg-mG2a was introduced by site-directed mutagenesis. Briefly, 293 suspension cells cultured in baffled flasks were diluted to 1.0 × 10⁶ cells/ml and co-transfected with 0.6 μg/ml of pFUSEss-CHIg-mG2a cloning vector containing VH of 9F4-WT or -K322A and 0.9 μg/ml of pFUSE2ss-CLIg-mK cloning vector containing VL gene of 9F4. pTT5 cloning vectors containing VH and VL of 9F4-LALA were also co-transfected as above. Antibodies expressed were purified using a HiTrap protein A affinity column. The column was eluted into fractions using 0.1 M glycine-HCl elution buffer (pH 2.7), and neutralized with sodium hydroxide. Fraction samples were analyzed using 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie brilliant blue staining. Fraction samples were pooled and dialysed in phosphate buffered saline (PBS) overnight at 4°C. Dialysed samples were filter sterilized using Ultrafree-CL centrifugal filters (Millipore) and quantified with Coomassie plus assay reagent (BioRad).

The VH and VL genes of 9F4 were cloned into pFUSEss-CHIg-mG2a and pFUSE2ss-CLIg-mK cloning vectors (InvivoGen) respectively in order to generate mouse IgG2a wild type 9F4. Amino acid substitution K322A in the fragment crystallisable region (Fc region) of 9F4-pFUSEss-CHIg-mG2a was introduced by site-directed mutagenesis. Briefly, 293 suspension cells cultured in baffled flasks were diluted to 1.0 × 10⁶ cells/ml and co-transfected with 0.6 μg/ml of pFUSEss-CHIg-mG2a cloning vector containing VH of 9F4-WT or -K322A and 0.9 μg/ml of pFUSE2ss-CLIg-mK cloning vector containing VL gene of 9F4. pTT5 cloning vectors containing VH and VL of 9F4-LALA were also co-transfected as above. Antibodies expressed were purified using a HiTrap protein A affinity column. The column was eluted into fractions using 0.1 M glycine-HCl elution buffer (pH 2.7), and neutralized with sodium hydroxide. Fraction samples were analyzed using 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie brilliant blue staining. Fraction samples were pooled and dialysed in phosphate buffered saline (PBS) overnight at 4°C. Dialysed samples were filter sterilized using Ultrafree-CL centrifugal filters (Millipore) and quantified with Coomassie plus assay reagent (BioRad).

THP-1 cells were lysed in buffer A (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, and 320 mM sucrose) supplemented with a protease inhibitor cocktail. Nuclei and unlysed cells were removed by centrifugation through 5 μm Ultrafree-CL centrifugal filters (Millipore). The filtrate was diluted with an equal volume of CHAPS buffer (20 mM HEPES-KOH, pH 7.5, 5 mM MgCl₂, 0.5 mM EGTA, and 0.1% CHAPS) supplemented with a protease inhibitor cocktail, and centrifuged to obtain the insoluble pelleted fraction. The pellets were resuspended in CHAPS buffer and then subjected to crosslinking using 2 mM disuccinimidyl suberate for 30 min. The protein samples were resolved by 12% SDS-PAGE, and the level of ASC was analyzed. ASC speck formation was analyzed in mouse BMDMs and ASC-mCherry-expressing THP-1 cells. ASC speck images were acquired under fluorescence microscopy (Olympus). For quantification, the ASC specks were counted with an IN Cell Analyzer (GE Healthcare) and normalized with respect to the number of nuclei, which were stained with 4′,6-diamidino-2-phenylindole.

THP-1 cells were lysed in buffer A (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, and 320 mM sucrose) supplemented with a protease inhibitor cocktail. The nuclei and unlysed cells were removed by centrifugation and 5 μm Ultrafree-CL centrifugal filters (Millipore). The filtrate was diluted with an equal volume of CHAPS buffer (20 mM HEPES-KOH, pH 7.5, 5 mM MgCl₂, 0.5 mM EGTA, and 0.1% CHAPS) supplemented with a protease inhibitor cocktail, and centrifuged to obtain insoluble pelleted fraction. The pellets were resuspended in CHAPS buffer and then subjected to cross-linking using disuccinimidyl suberate (DSS; 2 mM) for 30 min. The protein samples were resolved by 12% SDS-PAGE, and the level of ASC was analyzed. For the reconstitution of NLRP3 inflammasomes, HEK293T cells were transfected with pCMV-Flag-NLRP3, pLKO_AS2-ASC-Flag, and pLKO_AS2-ASC (Y146F)-Flag using Lipofectamine 2000. They were then incubated for 48 h and analyzed as described for THP-1 cells. ASC speck formation was analyzed in ASC-mCherry-expressing THP-1 cells. ASC speck images were acquired under fluorescence microscopy (Olympus). For quantification, the ASC speck was counted with an IN Cell Analyzer (GE Healthcare) and normalized with respect to the number of nuclei, which were stained with DAPI.

Nimotuzumab (5 mg/mL) in PBS was buffer exchanged in 0.1 M NaHCO₃ (pH 9) using centrifugal filters (Amicon Ultra-4 Centrifugal Filter 30K NMCO, EMD Millipore) and concentrated to 10 mg/mL nimotuzumab in the bicarbonate solution [21 (link)]. A ten-fold mole excess of p-SCN-Bz-Deferoxamine (DFO: Macrocyclics) in DMSO was added dropwise to the antibody solution (final volume of DMSO was kept below 5 %). The final concentration of the reaction mixture was 9.5 mg/mL. The reaction mixture was incubated at 37°C on a shaker at 650 RPM for an hour. The reaction mixture was cooled to room temperature and the unreacted DFO was removed by two consecutive centrifugations with desalting columns (Zeba™ Spin Desalting Columns, 7K MWCO, Thermofisher). The buffer was exchanged with PBS using centrifugation with centrifugal filters and sterilized with 0.2 μm hydrophilic PTFE membrane filters (Ultrafree®-CL Centrifugal filters, Millipore). The solution of DFO-nimotuzumab was aliquoted and stored at −80°C until further use.