ADAMTS13 siRNA

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	Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical veins as previously described (Tao et al., 2005) and cultured in M199 media supplemented with 20% fetal bovine serum penicillin, streptomycin, and amphotericin (Fungizone™). Upon reaching confluence, primary cultures of HUVEC were passaged using 0.25% trypsin/EDTA. Second-passage HUVEC were utilized in all assays. 24 h prior to the start of the knockdown assay, 2 × 105 first-passage HUVEC were added to a six-well plate in 2 ml of M199 media containing 20% FBS with antibiotics, such that approximately 60–80% confluence would be reached by the time of assay. The control and ADAMTS-13 siRNA knockdown assays were carried out according to the manufacturer's protocol. The cells or the cell lysates were harvested after 24 h incubation. ADAMTS-13 protein level in cell lysates from HUVEC treated with control siRNA or three different ADAMTS-13 siRNAs (s21868, s21869 and s21870). 15 nM siRNA was used for each transfection. After 24 h, cell lysates were harvested, concentrated and assayed with an ADAMTS-13 ELISA kit.

Protocol tips

Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical veins as previously described (Tao et al., 2005) and cultured in M199 media supplemented with 20% fetal bovine serum penicillin, streptomycin, and amphotericin (Fungizone™). Upon reaching confluence, primary cultures of HUVEC were passaged using 0.25% trypsin/EDTA. Second-passage HUVEC were utilized in all assays. 24 h prior to the start of the knockdown assay, 2 × 105 first-passage HUVEC were added to a six-well plate in 2 ml of M199 media containing 20% FBS with antibiotics, such that approximately 60–80% confluence would be reached by the time of assay. The control and ADAMTS-13 siRNA knockdown assays were carried out according to the manufacturer's protocol. The cells or the cell lysates were harvested after 24 h incubation. ADAMTS-13 protein level in cell lysates from HUVEC treated with control siRNA or three different ADAMTS-13 siRNAs (s21868, s21869 and s21870). 15 nM siRNA was used for each transfection. After 24 h, cell lysates were harvested, concentrated and assayed with an ADAMTS-13 ELISA kit.

Publication protocol

Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical veins as previously described (Tao et al., 2005) and cultured in M199 media supplemented with 20% fetal bovine serum penicillin, streptomycin, and amphotericin (Fungizone™). Upon reaching confluence, primary cultures of HUVEC were passaged using 0.25% trypsin/EDTA. Second-passage HUVEC were utilized in all assays. 24 h prior to the start of the knockdown assay, 2 × 105 first-passage HUVEC were added to a six-well plate in 2 ml of M199 media containing 20% FBS with antibiotics, such that approximately 60–80% confluence would be reached by the time of assay. The control and ADAMTS-13 siRNA knockdown assays were carried out according to the manufacturer's protocol. The cells or the cell lysates were harvested after 24 h incubation. ADAMTS-13 protein level in cell lysates from HUVEC treated with control siRNA or three different ADAMTS-13 siRNAs (s21868, s21869 and s21870). 15 nM siRNA was used for each transfection. After 24 h, cell lysates were harvested, concentrated and assayed with an ADAMTS-13 ELISA kit.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for ADAMTS13 siRNA below.

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