The model system was used as previously described, in which 1.0 MPa compression was loaded on NP cells to imitate in vivo condition [15 (link), 16 (link)]. The cells were treated with DMSO (Control, Sigma, USA), necroptosis inhibitor Necronstatin-1 (Nec-1, Sigma, USA), autophagy inhibitor 3-Methyladenine (3-MA, Sigma, USA), and apoptosis inhibitor Z-VAD-FMK (Z-VAD, Merck, Germany) and then using a combination of the inhibitors: Nec-1+3-MA and Nec-1+Z-VAD. The bottom of the pressure vessel was filled with distilled water to preserve sufficient humidity and keep the device in an incubator at 37°C. 0 h mentioned in the experiment means the beginning of compression. The 0, 24, and 36 h compression-treated time points were selected in the current experiment according to our previous studies [15 (link), 16 (link)].
Nec 1
Manufactured by Merck Group
Sourced in United States, Germany, Sao Tome and Principe, United Kingdom
Nec-1 is a laboratory instrument designed for the analysis and quantification of cellular apoptosis. It is an established tool for researchers studying programmed cell death mechanisms and their implications in various biological processes and disease states.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (12 mentions)
Z vad fmk, by Merck Group (9 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Z vad, by Merck Group (6 mentions)
Cycloheximide (chx), by Merck Group (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
N acetyl cysteine (nac), by Merck Group (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
N acetyl l cysteine, by Merck Group (4 mentions)
β actin, by Merck Group (3 mentions)
Fer 1, by Merck Group (3 mentions)
Z vad fmk zvad, by R&D Systems (3 mentions)
Flag tnfα, by Enzo Life Sciences (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Tnf α, by Thermo Fisher Scientific (3 mentions)
Q vd oph, by Merck Group (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Z vad fmk, by Selleck Chemicals (3 mentions)
Z vad fmk, by R&D Systems (2 mentions)
Gsk 872, by Merck Group (2 mentions)
101 protocols using nec 1
1
Mechanobiology of Nucleus Pulposus Cells
2
Hemorrhage/Trauma-Induced MODS Study
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The experimental rats were randomly divided into three groups: (1) the sham group underwent the same anesthetic and surgical procedures and fluid resuscitation without induction of hemorrhage/trauma (n = 25); (2) the MODS group was described above (n = 25); and (3) the Nec-1 treatment group (n = 25), and the Nec-1 was purchased by Sigma, MO, USA, in which the MODS rats were pretreatment and once daily with Nec-1 (250 μg), which was diluted in resuscitation fluid and administered through the right jugular vein for about 20 minutes at a constant rate via a mini-pump. The dose of Nec-1 used in this study was based on previous study [11 (link)].
3
Investigating Cell Death Pathways
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The following reagents were used: zVAD-FMK (10 μM, Apex Bio), QVD (10 μM, Apex Bio), SM-164 (100 nM, gift from Shaomeng Wang), GSK’963 (referred as RIPK1i, 100 nM, GlaxoSmithKline), Nec-1 (10 μM Merck), Nec-1s (10 μM Merck), RO-3306 (9 μM, Merck), Thymidine (Sigma), BIM peptide (Kind gift of Tony Letai), FLAG-hTNF (10 ng/ml, Enzo), MG132 (1-20 μM, as indicated per cell line, see below, SIGMA), Recombinant Casp8 (1U Enzo). The following antibodies were used for western blotting: α-RIPK1 (1:1000, BD Biosciences), α-HA (1:1000, Roche), α-PLK1 (1:1000, Bethyl Laboratories), α-PLK1-pT210 (1:2000, AbCam), α-Cyclin B (1:1000, Cell Signaling), α-BUBR1 (1:1000, BD Bioscience), α-BUBR1-pT680 (1:1000, Kind gift of Geert J.P.L Kops), α-BUBR1-pT676 (1:1000, Kind gift of Erich A. Nigg), α-pH-H3 (1:2000, Millipore), α-Casp8 (for WB - post IP, 1:5000, MBL), α-Casp8 - for IP (7.5 μg/ml, C-20, Santa Cruz Biotechnology, α-FADD – for IP (7.5 μg/ml, Santa Cruz), α-FADD (1:1000 BD Biosciences), α-RIPK3 (1:1000 Proscience), α-RIPK3 (1:1000 Novus Biological) α-cFLIP (1:1000, Enzo), α-Myc (1:1000, clone 9E10,SIGMA), α-HSP90 (1:1000 Santa Cruz Biotechnology).
4
Intracerebroventricular Administration of Nec-1
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Nec-1 (25 mg, Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) to a final concentration of 40 µg/µL. Intracerebroventricular administration was performed as described previously.20 (link) A single dose of Nec-1 (5 µL) was administered using a 10 µL syringe 30 min after the surgery. The detailed process was similar to that described by others (coordinates: 1.0 mm posterior, 1.8 mm lateral to bregma, 4 mm depth).21 (link) The sham and vehicle group received an equal volume of DMSO in the same way.
5
Nec-1 Protects Against IVH-Induced Injury
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Nec-1 (Selleck; Houston, TX, USA) was diluted with dimethyl sulfoxide (Sigma) to a concentration of 2.6 μg/μL. For the IVH + Nec-1 group, 1 μL Nec-1 (2.6 μg) solution was administered into the single lateral ventricle (co-ordinates relative to the bregma: 0.5 mm anterior to the bregma, 1.5 mm lateral of the bregma, and 2.2 mm in depth) one hour prior to IVH induction. Animals that died were excluded from the study.
6
Leishmania infantum Infection of Neutrophils
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Neutrophils were infected in vitro with L. infantum promastigotes stationary-phase at a ratio of 1:2 (neutrophil:parasites). For assays of cell death, mouse and human neutrophils were pretreated for 30 min with zVAD-fmk (100 µM) (R&D Systems, Minneapolis, MN, USA) or zIETD-fmk (100 µM) (R&D Systems, Minneapolis, MN, USA) to block caspase activation before infection. In some experiments, Nec-1 (50 µM), GSK’872 (3 µM), or NSA (10 µM), necroptosis inhibitors (all from Merck Millipore’s Calbiochem®, Darmstadt, Germany) were used. DMSO (vehicle) 0.4% (Cayman Chemical; Ann Arbor, MI, USA) was used as control. After 18 h, mouse infected neutrophils, or after 3 h, human infected neutrophils, were centrifuged, supernatants containing noninternalized promastigotes were collected, and medium was replaced by 250 µl Schneider insect medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 20% inactive FBS, 2 mM l -glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin. After that, infected neutrophils were cultured at 25°C for an additional 3 days and intracellular load of L. infantum was estimated by production of proliferating extracellular motile promastigotes in Schneider medium (43 (link)).
7
RIPK1-Mediated Necroptosis Signaling
8
Cell Death Induction and Inhibition
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Chemicals were obtained from Sigma unless otherwise indicated. Cells were treated with either zVad (25 μM or 50 μM, Bachem), Necrostatin-1 (Nec-1, 50 μM), caspase-8 inhibitor (50 μM, Merck), or respective combinations 30 min prior stimulation with either DMF, Etoposide (Biovision), LCL161 (Active Biochem), NBDII (Merck), PMX464 (Tocris) or TNFα (10 ng/ml) and Cycloheximide (Chx, 10 μg/ml). Cells were incubated with 5 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) 10 min before measurement.
9
Nec-1 Attenuates LPS-Induced Acute Lung Injury in Mice
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All animal procedures were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University. Mice were fasted for 8 h prior to the experiment. Before induction of ALI, the mice were anesthetized by inhalation of sevoflurane and were transtracheally injected with either Nec-1 (5 mg/kg; Enzo Life Sciences, Inc.) or saline as a control. LPS-induced ALI was induced by transtracheal injection of LPS (Sigma-Aldrich; Merck KGaA) at a dose of 10 mg/kg 30 min after Nec-1 or saline treatment. Additionally, the mice of sham-operation were transtracheally injected with saline and treated with saline only. During the experiment, the mice were monitored every 30 min. All mice survived until sacrifice 6 h after initiation of the experiment. The mice were sacrificed by cervical dislocation under 3% sevoflurane inhalation, after which, the lungs were quickly removed. Lung samples were either placed in a buffered solution for histopathological analysis, or snap-frozen for protein isolation or for analyzing apoptosis by flow cytometry. Prior to tissue collection, death was confirmed after 1–2 min of cardiac and respiratory observation.
10
Preparation of PMA and Nec-1 Solutions
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Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, USA, Cat No.) at an original concentration of 1 mg/mL was diluted with phosphate-buffered saline (PBS, Thermo Fisher, USA, Cat No.10010023) to a working solution of 1 μg/mL. Nec-1 (Merck, Germany, Cat No. 480065) at an original concentration of 5 mg/mL was diluted with PBS to a working solution of 1 mg/mL.
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