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Avantor leupeptin
Inhibitors of v-ATPase are the most potent activators of MTORC1 in chondrocytes. ( A ) MEFs and C5.18 chondrocytes were treated with varying doses of concanamycin for 48 h. ( B ) C5.18 chondrocytes were treated with varying doses of Baf, 3-MA, CQ and <t>leupeptin</t> for 48 h. In all panels the levels of p-RPS6 and SQSTM1 were analyzed by western blot.
Leupeptin, supplied by Avantor, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner"

Article Title: Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

Journal: Autophagy

doi: 10.1080/15548627.2015.1068489

Inhibitors of v-ATPase are the most potent activators of MTORC1 in chondrocytes. ( A ) MEFs and C5.18 chondrocytes were treated with varying doses of concanamycin for 48 h. ( B ) C5.18 chondrocytes were treated with varying doses of Baf, 3-MA, CQ and leupeptin for 48 h. In all panels the levels of p-RPS6 and SQSTM1 were analyzed by western blot.
Figure Legend Snippet: Inhibitors of v-ATPase are the most potent activators of MTORC1 in chondrocytes. ( A ) MEFs and C5.18 chondrocytes were treated with varying doses of concanamycin for 48 h. ( B ) C5.18 chondrocytes were treated with varying doses of Baf, 3-MA, CQ and leupeptin for 48 h. In all panels the levels of p-RPS6 and SQSTM1 were analyzed by western blot.

Techniques Used: Western Blot

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Transferring:

Article Title: Identification of brain antigens recognized by autoantibodies in experimental autoimmune encephalomyelitis-induced animals treated with etomoxir or interferon-β
Article Snippet: .. Preparation of rat brain sample Cerebellum from three-week-old Sprague-Dawley rats was isolated, and protein homogenates were prepared by transferring the cerebellum to a dissection buffer containing 10% sucrose (VWR), imidazole (Merck Millipore), EDTA (Sigma-Aldrich), Pefabloc (Sigma-Aldrich) and leupeptin (VWR). .. The mixture was homogenized (T10 basic ULTRA-TURAX homogenizer IKA®) for 20 sec; then, samples were centrifuged at 1000 g for 15 min. Supernatants were stored at −20 °C.

Article Title: Identification of brain antigens recognized by autoantibodies in experimental autoimmune encephalomyelitis-induced animals treated with etomoxir or interferon-β
Article Snippet: .. Cerebellum from three-week-old Sprague-Dawley rats was isolated, and protein homogenates were prepared by transferring the cerebellum to a dissection buffer containing 10% sucrose (VWR), imidazole (Merck Millipore), EDTA (Sigma-Aldrich), Pefabloc (Sigma-Aldrich) and leupeptin (VWR). .. The mixture was homogenized (T10 basic ULTRA-TURAX homogenizer IKA®) for 20 sec; then, samples were centrifuged at 1000 g for 15 min. Supernatants were stored at −20 °C.

Homogenization:

Article Title: Nicotiana plumbaginifolia plants silenced for the ATP‐binding cassette transporter gene NpPDR1 show increased susceptibility to a group of fungal and oomycete pathogens
Article Snippet: .. Plant material was ground in 2 vol of cold homogenization buffer [250 m m sorbitol, 50 m m Tris‐HCl, pH 8.0, 2 m m ethylenediaminetetraacetic acid (EDTA), 6 g/L polyvinylpyrrolidone, 5 m m dithiothreitol (DTT), 1 m m phenylmethylsulphonylfluoride (PMSF) and 2 µg/mL each of leupeptin, pepstatin, aprotinin, antipain and chymostatin] using a 1‐mL glass ‘Duall’ grinder (VWR). .. Cell debris was removed by centrifugation for 5 min at 3500 g and the supernatant was centrifuged for 5 min at 10 000 g .

Isolation:

Article Title: Identification of brain antigens recognized by autoantibodies in experimental autoimmune encephalomyelitis-induced animals treated with etomoxir or interferon-β
Article Snippet: .. Preparation of rat brain sample Cerebellum from three-week-old Sprague-Dawley rats was isolated, and protein homogenates were prepared by transferring the cerebellum to a dissection buffer containing 10% sucrose (VWR), imidazole (Merck Millipore), EDTA (Sigma-Aldrich), Pefabloc (Sigma-Aldrich) and leupeptin (VWR). .. The mixture was homogenized (T10 basic ULTRA-TURAX homogenizer IKA®) for 20 sec; then, samples were centrifuged at 1000 g for 15 min. Supernatants were stored at −20 °C.

Article Title: Identification of brain antigens recognized by autoantibodies in experimental autoimmune encephalomyelitis-induced animals treated with etomoxir or interferon-β
Article Snippet: .. Cerebellum from three-week-old Sprague-Dawley rats was isolated, and protein homogenates were prepared by transferring the cerebellum to a dissection buffer containing 10% sucrose (VWR), imidazole (Merck Millipore), EDTA (Sigma-Aldrich), Pefabloc (Sigma-Aldrich) and leupeptin (VWR). .. The mixture was homogenized (T10 basic ULTRA-TURAX homogenizer IKA®) for 20 sec; then, samples were centrifuged at 1000 g for 15 min. Supernatants were stored at −20 °C.

Dissection:

Article Title: Identification of brain antigens recognized by autoantibodies in experimental autoimmune encephalomyelitis-induced animals treated with etomoxir or interferon-β
Article Snippet: .. Preparation of rat brain sample Cerebellum from three-week-old Sprague-Dawley rats was isolated, and protein homogenates were prepared by transferring the cerebellum to a dissection buffer containing 10% sucrose (VWR), imidazole (Merck Millipore), EDTA (Sigma-Aldrich), Pefabloc (Sigma-Aldrich) and leupeptin (VWR). .. The mixture was homogenized (T10 basic ULTRA-TURAX homogenizer IKA®) for 20 sec; then, samples were centrifuged at 1000 g for 15 min. Supernatants were stored at −20 °C.

Article Title: Identification of brain antigens recognized by autoantibodies in experimental autoimmune encephalomyelitis-induced animals treated with etomoxir or interferon-β
Article Snippet: .. Cerebellum from three-week-old Sprague-Dawley rats was isolated, and protein homogenates were prepared by transferring the cerebellum to a dissection buffer containing 10% sucrose (VWR), imidazole (Merck Millipore), EDTA (Sigma-Aldrich), Pefabloc (Sigma-Aldrich) and leupeptin (VWR). .. The mixture was homogenized (T10 basic ULTRA-TURAX homogenizer IKA®) for 20 sec; then, samples were centrifuged at 1000 g for 15 min. Supernatants were stored at −20 °C.

Western Blot:

Article Title: Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner
Article Snippet: .. Materials and Methods The following chemicals and reagents were used in the preparation of this manuscript: Phosphate buffered saline (PBS; Invitrogen, 1160381), Dulbecco's modified Eagle's medium/F-12 mixture (DMEM/F12; Invitrogen, 11330057), DMEM custom medium (made by Life Technologies and is a standard DMEM medium depleted of all amino acids, glucose and pyruvate), minimal essential medium (Invitrogen, 22571020), fetal bovine serum (FBS; Life Technologies, 16000-044), gentamycin (Invitrogen, 15750), ascorbic acid (Sigma-Aldrich, A5960), β-glycerophosphate (βGP; Sigma-Aldrich, G9422), doxycycline (dox; Sigma-Aldrich, D3447), bafilomycin A1 (LC laboratories, B-1080), concanamycin A (Sigma-Aldrich, 27689), chloroquine (Sigma-Aldrich, C6628), dexamethasone (Sigma-Aldrich, D1756), Torin1 (Tocris Biosciences, 4247), hyaluronidase (Sigma-Aldrich, H3506), leupeptin (VWR, a2183.0025), ECL western blotting substrate (Perkin Elmer, NEL104001EA), 2x Laemmli buffer (Sigma-Aldrich, S3401), alcian blue (Sigma-Aldrich, A5268), digoxigenin (Roche Inc., 11277073910), bovine serum albumin (Sigma-Aldrich, A8806), IGF1 (Sigma-Aldrich, I3769), Magic Cathepsin B kit (ImmunoChemistry Technologies, 937), rat-tail collagen (Invitrogen, A1048301), microscope cover glasses (VWR International, ECN 631-1578), 3-methyladenine (Sigma-Aldrich, M9281), Vectastatin avidin-biotin complex (ABC) kit (Vector laboratories, PK-6100), 3, 3′-diaminobenzidine kit (DAKO, K3468), and normal horse serum (DAKO, S-2000). .. The following antibodies were used: anti-p-RPS6 (Cell Signaling Technology, 4858), anti-AKT (Cell Signaling Technology, 9272), anti-p-AKT (Ser473; Cell Signaling Technology, 4060), anti-RPS6 (5G10; Cell Signaling Technology, 2217), anti-GAPDH (Cell Signaling Technology, 2118), anti-EIF4EBP1 (53H11; Cell Signaling Technology, 9644), anti-phospho-EIF4EBP1 (Thr37/46; Cell Signaling Technology, 2855), anti-MAP1LC3A (MBL, PM036), LAMP2 (Santa Cruz Biotechnology, sc-8100), FLCN (Cell Signaling Technology, 3697), anti-ATG5 (Cell Signaling Technology, 8540), anti-SQSTM1 (Progen Bioteknik, GP62-C), anti-COL10A1 (Quartett; GMBH, 1-CO097-05), biotin-conjugated anti-guinea pig, anti-rabbit and anti-mouse secondary antibodies (Jackson ImmunoResearch,106-065-003, 111-066-003 and 115-066-003, respectively), mouse anti-BrdU antibody (Sigma, B8434), biotinylated anti-mouse antibody (Jackson, 115-066-003), streptavidin-conjugated Alexa Fluor 546 (Life Technologies, S11225), fluorescently labeled anti-goat antibody (Jackson, 705165147) and fluorescently labeled anti-rabbit antibody (Jackson, 711545152).

Avidin-Biotin Assay:

Article Title: Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner
Article Snippet: .. Materials and Methods The following chemicals and reagents were used in the preparation of this manuscript: Phosphate buffered saline (PBS; Invitrogen, 1160381), Dulbecco's modified Eagle's medium/F-12 mixture (DMEM/F12; Invitrogen, 11330057), DMEM custom medium (made by Life Technologies and is a standard DMEM medium depleted of all amino acids, glucose and pyruvate), minimal essential medium (Invitrogen, 22571020), fetal bovine serum (FBS; Life Technologies, 16000-044), gentamycin (Invitrogen, 15750), ascorbic acid (Sigma-Aldrich, A5960), β-glycerophosphate (βGP; Sigma-Aldrich, G9422), doxycycline (dox; Sigma-Aldrich, D3447), bafilomycin A1 (LC laboratories, B-1080), concanamycin A (Sigma-Aldrich, 27689), chloroquine (Sigma-Aldrich, C6628), dexamethasone (Sigma-Aldrich, D1756), Torin1 (Tocris Biosciences, 4247), hyaluronidase (Sigma-Aldrich, H3506), leupeptin (VWR, a2183.0025), ECL western blotting substrate (Perkin Elmer, NEL104001EA), 2x Laemmli buffer (Sigma-Aldrich, S3401), alcian blue (Sigma-Aldrich, A5268), digoxigenin (Roche Inc., 11277073910), bovine serum albumin (Sigma-Aldrich, A8806), IGF1 (Sigma-Aldrich, I3769), Magic Cathepsin B kit (ImmunoChemistry Technologies, 937), rat-tail collagen (Invitrogen, A1048301), microscope cover glasses (VWR International, ECN 631-1578), 3-methyladenine (Sigma-Aldrich, M9281), Vectastatin avidin-biotin complex (ABC) kit (Vector laboratories, PK-6100), 3, 3′-diaminobenzidine kit (DAKO, K3468), and normal horse serum (DAKO, S-2000). .. The following antibodies were used: anti-p-RPS6 (Cell Signaling Technology, 4858), anti-AKT (Cell Signaling Technology, 9272), anti-p-AKT (Ser473; Cell Signaling Technology, 4060), anti-RPS6 (5G10; Cell Signaling Technology, 2217), anti-GAPDH (Cell Signaling Technology, 2118), anti-EIF4EBP1 (53H11; Cell Signaling Technology, 9644), anti-phospho-EIF4EBP1 (Thr37/46; Cell Signaling Technology, 2855), anti-MAP1LC3A (MBL, PM036), LAMP2 (Santa Cruz Biotechnology, sc-8100), FLCN (Cell Signaling Technology, 3697), anti-ATG5 (Cell Signaling Technology, 8540), anti-SQSTM1 (Progen Bioteknik, GP62-C), anti-COL10A1 (Quartett; GMBH, 1-CO097-05), biotin-conjugated anti-guinea pig, anti-rabbit and anti-mouse secondary antibodies (Jackson ImmunoResearch,106-065-003, 111-066-003 and 115-066-003, respectively), mouse anti-BrdU antibody (Sigma, B8434), biotinylated anti-mouse antibody (Jackson, 115-066-003), streptavidin-conjugated Alexa Fluor 546 (Life Technologies, S11225), fluorescently labeled anti-goat antibody (Jackson, 705165147) and fluorescently labeled anti-rabbit antibody (Jackson, 711545152).

Microscopy:

Article Title: Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner
Article Snippet: .. Materials and Methods The following chemicals and reagents were used in the preparation of this manuscript: Phosphate buffered saline (PBS; Invitrogen, 1160381), Dulbecco's modified Eagle's medium/F-12 mixture (DMEM/F12; Invitrogen, 11330057), DMEM custom medium (made by Life Technologies and is a standard DMEM medium depleted of all amino acids, glucose and pyruvate), minimal essential medium (Invitrogen, 22571020), fetal bovine serum (FBS; Life Technologies, 16000-044), gentamycin (Invitrogen, 15750), ascorbic acid (Sigma-Aldrich, A5960), β-glycerophosphate (βGP; Sigma-Aldrich, G9422), doxycycline (dox; Sigma-Aldrich, D3447), bafilomycin A1 (LC laboratories, B-1080), concanamycin A (Sigma-Aldrich, 27689), chloroquine (Sigma-Aldrich, C6628), dexamethasone (Sigma-Aldrich, D1756), Torin1 (Tocris Biosciences, 4247), hyaluronidase (Sigma-Aldrich, H3506), leupeptin (VWR, a2183.0025), ECL western blotting substrate (Perkin Elmer, NEL104001EA), 2x Laemmli buffer (Sigma-Aldrich, S3401), alcian blue (Sigma-Aldrich, A5268), digoxigenin (Roche Inc., 11277073910), bovine serum albumin (Sigma-Aldrich, A8806), IGF1 (Sigma-Aldrich, I3769), Magic Cathepsin B kit (ImmunoChemistry Technologies, 937), rat-tail collagen (Invitrogen, A1048301), microscope cover glasses (VWR International, ECN 631-1578), 3-methyladenine (Sigma-Aldrich, M9281), Vectastatin avidin-biotin complex (ABC) kit (Vector laboratories, PK-6100), 3, 3′-diaminobenzidine kit (DAKO, K3468), and normal horse serum (DAKO, S-2000). .. The following antibodies were used: anti-p-RPS6 (Cell Signaling Technology, 4858), anti-AKT (Cell Signaling Technology, 9272), anti-p-AKT (Ser473; Cell Signaling Technology, 4060), anti-RPS6 (5G10; Cell Signaling Technology, 2217), anti-GAPDH (Cell Signaling Technology, 2118), anti-EIF4EBP1 (53H11; Cell Signaling Technology, 9644), anti-phospho-EIF4EBP1 (Thr37/46; Cell Signaling Technology, 2855), anti-MAP1LC3A (MBL, PM036), LAMP2 (Santa Cruz Biotechnology, sc-8100), FLCN (Cell Signaling Technology, 3697), anti-ATG5 (Cell Signaling Technology, 8540), anti-SQSTM1 (Progen Bioteknik, GP62-C), anti-COL10A1 (Quartett; GMBH, 1-CO097-05), biotin-conjugated anti-guinea pig, anti-rabbit and anti-mouse secondary antibodies (Jackson ImmunoResearch,106-065-003, 111-066-003 and 115-066-003, respectively), mouse anti-BrdU antibody (Sigma, B8434), biotinylated anti-mouse antibody (Jackson, 115-066-003), streptavidin-conjugated Alexa Fluor 546 (Life Technologies, S11225), fluorescently labeled anti-goat antibody (Jackson, 705165147) and fluorescently labeled anti-rabbit antibody (Jackson, 711545152).

Incubation:

Article Title: Role of Src and Cortactin in Pemphigus Skin Blistering
Article Snippet: .. Triton X-100 Protein Fractionation Cells were washed with ice-cold PBS and incubated in Triton buffer (0.5% Triton X-100, 50 mM MES, 25 mM EGTA, 5 mM MgCl2 ) supplemented with 1 mM PMSF (Roth, Germany), Aprotinin, Pepstatin A (both Applichem, Germany), and Leupeptin (VWR, Germany) for 20 min on ice under continuous shaking. ..

Article Title: NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways
Article Snippet: .. To obtain neutrophil lysate, cell pellets were resuspended in a lysis buffer [1% (v/v) Triton X-100, 50 mM Tris, pH 7.4, 10 mM KCl containing 2× complete, mini protease inhibitor cocktail (Roche Diagnostics, Laval, QC, Canada) supplemented with 0.5 mM EDTA, 25 μM leupeptin, 25 μM pepstatin, 25 μM aprotinin, 1 mM levamisole, 1 mM Na3 VO4 , 25 mM NaF, 1 mM PMSF], sonicated (VWR Sonics model 50D), and incubated for 15 min at 4°C. ..

Fractionation:

Article Title: Role of Src and Cortactin in Pemphigus Skin Blistering
Article Snippet: .. Triton X-100 Protein Fractionation Cells were washed with ice-cold PBS and incubated in Triton buffer (0.5% Triton X-100, 50 mM MES, 25 mM EGTA, 5 mM MgCl2 ) supplemented with 1 mM PMSF (Roth, Germany), Aprotinin, Pepstatin A (both Applichem, Germany), and Leupeptin (VWR, Germany) for 20 min on ice under continuous shaking. ..

Expressing:

Article Title: Potential adverse effects of botanical supplementation in high-fat-fed female mice
Article Snippet: .. Analysis of protein expression Skeletal muscle and liver lysates were prepared from powdered tissue by homogenizing in 25 mM HEPES, pH 7.4, 1% Igepal CA630, 137 mM NaCl, 1 mM PMSF, 10 μg/ml aprotinin, 1 μg/ml pepstatin, 5 μg/ml leupeptin, 10 mM Na4 P2 O7 , 100 mM NaF, and 2 mM NaVO4 using a Sonifier 450 homogenizer (VWR, Radnor, PA). .. Protein concentrations were determined using a BCA assay (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s instructions.

Lysis:

Article Title: NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways
Article Snippet: .. To obtain neutrophil lysate, cell pellets were resuspended in a lysis buffer [1% (v/v) Triton X-100, 50 mM Tris, pH 7.4, 10 mM KCl containing 2× complete, mini protease inhibitor cocktail (Roche Diagnostics, Laval, QC, Canada) supplemented with 0.5 mM EDTA, 25 μM leupeptin, 25 μM pepstatin, 25 μM aprotinin, 1 mM levamisole, 1 mM Na3 VO4 , 25 mM NaF, 1 mM PMSF], sonicated (VWR Sonics model 50D), and incubated for 15 min at 4°C. ..

Modification:

Article Title: Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner
Article Snippet: .. Materials and Methods The following chemicals and reagents were used in the preparation of this manuscript: Phosphate buffered saline (PBS; Invitrogen, 1160381), Dulbecco's modified Eagle's medium/F-12 mixture (DMEM/F12; Invitrogen, 11330057), DMEM custom medium (made by Life Technologies and is a standard DMEM medium depleted of all amino acids, glucose and pyruvate), minimal essential medium (Invitrogen, 22571020), fetal bovine serum (FBS; Life Technologies, 16000-044), gentamycin (Invitrogen, 15750), ascorbic acid (Sigma-Aldrich, A5960), β-glycerophosphate (βGP; Sigma-Aldrich, G9422), doxycycline (dox; Sigma-Aldrich, D3447), bafilomycin A1 (LC laboratories, B-1080), concanamycin A (Sigma-Aldrich, 27689), chloroquine (Sigma-Aldrich, C6628), dexamethasone (Sigma-Aldrich, D1756), Torin1 (Tocris Biosciences, 4247), hyaluronidase (Sigma-Aldrich, H3506), leupeptin (VWR, a2183.0025), ECL western blotting substrate (Perkin Elmer, NEL104001EA), 2x Laemmli buffer (Sigma-Aldrich, S3401), alcian blue (Sigma-Aldrich, A5268), digoxigenin (Roche Inc., 11277073910), bovine serum albumin (Sigma-Aldrich, A8806), IGF1 (Sigma-Aldrich, I3769), Magic Cathepsin B kit (ImmunoChemistry Technologies, 937), rat-tail collagen (Invitrogen, A1048301), microscope cover glasses (VWR International, ECN 631-1578), 3-methyladenine (Sigma-Aldrich, M9281), Vectastatin avidin-biotin complex (ABC) kit (Vector laboratories, PK-6100), 3, 3′-diaminobenzidine kit (DAKO, K3468), and normal horse serum (DAKO, S-2000). .. The following antibodies were used: anti-p-RPS6 (Cell Signaling Technology, 4858), anti-AKT (Cell Signaling Technology, 9272), anti-p-AKT (Ser473; Cell Signaling Technology, 4060), anti-RPS6 (5G10; Cell Signaling Technology, 2217), anti-GAPDH (Cell Signaling Technology, 2118), anti-EIF4EBP1 (53H11; Cell Signaling Technology, 9644), anti-phospho-EIF4EBP1 (Thr37/46; Cell Signaling Technology, 2855), anti-MAP1LC3A (MBL, PM036), LAMP2 (Santa Cruz Biotechnology, sc-8100), FLCN (Cell Signaling Technology, 3697), anti-ATG5 (Cell Signaling Technology, 8540), anti-SQSTM1 (Progen Bioteknik, GP62-C), anti-COL10A1 (Quartett; GMBH, 1-CO097-05), biotin-conjugated anti-guinea pig, anti-rabbit and anti-mouse secondary antibodies (Jackson ImmunoResearch,106-065-003, 111-066-003 and 115-066-003, respectively), mouse anti-BrdU antibody (Sigma, B8434), biotinylated anti-mouse antibody (Jackson, 115-066-003), streptavidin-conjugated Alexa Fluor 546 (Life Technologies, S11225), fluorescently labeled anti-goat antibody (Jackson, 705165147) and fluorescently labeled anti-rabbit antibody (Jackson, 711545152).

Sonication:

Article Title: NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways
Article Snippet: .. To obtain neutrophil lysate, cell pellets were resuspended in a lysis buffer [1% (v/v) Triton X-100, 50 mM Tris, pH 7.4, 10 mM KCl containing 2× complete, mini protease inhibitor cocktail (Roche Diagnostics, Laval, QC, Canada) supplemented with 0.5 mM EDTA, 25 μM leupeptin, 25 μM pepstatin, 25 μM aprotinin, 1 mM levamisole, 1 mM Na3 VO4 , 25 mM NaF, 1 mM PMSF], sonicated (VWR Sonics model 50D), and incubated for 15 min at 4°C. ..

Protease Inhibitor:

Article Title: NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways
Article Snippet: .. To obtain neutrophil lysate, cell pellets were resuspended in a lysis buffer [1% (v/v) Triton X-100, 50 mM Tris, pH 7.4, 10 mM KCl containing 2× complete, mini protease inhibitor cocktail (Roche Diagnostics, Laval, QC, Canada) supplemented with 0.5 mM EDTA, 25 μM leupeptin, 25 μM pepstatin, 25 μM aprotinin, 1 mM levamisole, 1 mM Na3 VO4 , 25 mM NaF, 1 mM PMSF], sonicated (VWR Sonics model 50D), and incubated for 15 min at 4°C. ..

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