293FT cells (Invitrogen) were transfected with expression vectors for wild-type or mutant NHLRC2-HA using polyethyleneimine “MAX” transfection reagent (Polysciences, Warrington, PA, USA) and then lysed in Cell Lysis Buffer (BioVision, Milpitas, CA, USA). Aliquots of lysates were incubated with 1 unit of recombinant active caspases (BioVision) in 1 × Reaction Buffer (BioVision) at 37 °C for 1 h. Reactions were terminated by adding Laemmli sample buffer and boiling at 95 °C for 5 min. Cleavage was evaluated by immunoblotting using an anti-NHLRC2 antibody.
Cell lysis buffer
Manufactured by Abcam
Sourced in United States, United Kingdom
Cell lysis buffer is a solution used to disrupt the cell membrane and release the cellular contents, including proteins, nucleic acids, and other biomolecules, for further analysis or purification.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Goat anti mouse igg hrp, by Proteintech (3 mentions)
Hrp goat anti rabbit igg, by Proteintech (3 mentions)
Mouse anti gapdh, by Proteintech (3 mentions)
Enhanced chemiluminescent reagent, by Thermo Fisher Scientific (3 mentions)
Mini protean tgx gel, by Bio-Rad (3 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Megafasligand, by Adipogen (2 mentions)
Genepix pro 7, by Molecular Devices (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ab181602, by Abcam (2 mentions)
Lmaxii 384 luminometer, by Molecular Devices (2 mentions)
Caspase 3 colorimetric assay kit, by Abcam (2 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Cathepsin b activity fluorometric assay kit, by Abcam (2 mentions)
Trans blot turbo transfer system and reagents, by Bio-Rad (2 mentions)
G box f3 imaging system, by Syngene (2 mentions)
Image lab analysis software, by Bio-Rad (2 mentions)
53 protocols using cell lysis buffer
1
NHLRC2 Cleavage by Caspases
2
FasL-Induced T Cell Apoptosis Assay
3
Antibody Array Analysis of CR and NR EVs
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For antibody array, CTB- and AV-EVs isolated from CR and NR plasma pools were lysed with cell lysis buffer (#K269; Biovision) and 100 μL of the protein lysate were analyzed using the Fullmoon Biosystems Explorador Antibody (#ASB600, Fullmoon Biosystems) according to manufacturer’s instructions. We also conducted the same analysis with 100 μL of crude plasma (without EVs isolation) from both patient pools. After the immune reaction, following the manufacturer’s recommendations, the arrays were scanned and the values were normalized using GenePix Pro 7 software (Molecular Devices) to correct for any technical, chip-to-chip, or day-to-day variations. Since in the matrix there were two replicates of each spot, the relative expression means between the replicates were calculated. The reactivity against the controls contained in each matrix was used as background cutoff, and the reactivity higher than the background was classified as present and the lower reactivity as absent.
4
FasL-Induced T Cell Apoptosis Assay
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Primary human T cells were stimulated for 3 days with anti-CD3 and anti-CD28, and the cells were then washed and cultured in complete RPMI with IL2 (10 µg/mL) for 4 additional days. For western blot analysis, 10 mL of stimulated primary human T cells (1.5 million cells/mL) in RPMI with IL2 were then treated with the indicated compounds for 1h prior to addition of FasL (1 µL of 100 µg/µL stock solution of MegaFasLigand™ in water, final concentration =10 ng/mL, Adipogen). After 3 hours, cells were harvested by centrifugation, washed in PBS and lysed in cell lysis buffer (BioVision, 1067-100) with 1 × cOmplete protease inhibitor (Roche) and 40 µg of each sample were separated by SDS-Page on 14% polyacrylamide gels. The gels were transferred to nitrocellulose membranes and were immunoblotted overnight with the indicated antibodies. For measurements of cell viability, in triplicate for each condition, 150,000 cells (100 µL of 1.5 million cells/mL ) were plated in 96-Well Optical-Bottom Plates. FasL was used at the same concentration indicated above with a 30 minute pre-incubation with compounds at the indicated concentrations, followed by 4 hours with FasL or DMSO. 20 × compound stock solutions were made in RPMI immediately before use. Cell viability was measured with CellTiter-Glo® Luminescent Cell Viability Assay (Promega) and was read on a Biotech Synergy 4 plate reader.
5
Syndecan-1 Levels in Sepsis Model
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Animals were injected IP with 50 µL saline, 125 µg S. aureus LTA or 500 ng TNFα, n = 5. Four hours after, the mice were anaesthetized with a subcutaneous injection of a mixture of ketamine and xylazine. Peritoneal lavage was done with 2 mL of clinical grade saline. Blood was collected via cardiac puncture in a heparinized syringe. Peritoneal effluent and blood were centrifuged at 1188×g. Samples of the abdominal wall were collected and immediately frozen in liquid nitrogen. The tissues were homogenized in cell lysis buffer (BioVision, Milpitas, CA, USA). Blood plasma and the supernatant from the tissue homogenate were analyzed at a 50-fold dilution. Peritoneal effluent was analyzed at a 10-fold dilution in PBS. Syndecan-1 levels in the tissue homogenate, peritoneal effluent and blood plasma were measured with a commercially available ELISA kit for mouse syndecan-1 (E91966Mu; USCNK Life Sciences Inc., Wuhan, China). To account for differences in tissue sample sizes, a Bradford assay was performed to measure total protein concentration in tissue homogenates and results were normalized to the total protein recovered.
6
Proteasome Activity Fluorescence Assay
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After being treated with I-Trp for designated time periods, cells were collected and resuspended in cell lysis buffer (BioVision). Aliquots of cell lysates (100 μg) were incubated with 1 μM of fluorescent proteasome substrate (BioVision) for 30 min at 37 °C. The fluorescent intensity was read using a fluorimeter with an exciting wavelength at 355 nm and emission wavelength at 405 mm and presented as relative proteasome activity.
7
Cell Lysis and Immunoblot Analysis
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Cells were treated with cell lysis buffer (Biovision, Milpitas, CA) including a protease inhibitor cocktail (Roche) for 20 min on ice. Lysed cells were centrifuged for 20 min at 12,000 rcf and supernatant was collected and frozen at −80°C until final analysis. An appropriate amount of protein was applied per lane for immunoblot analysis. Free binding sites of the nitrocellulose membrane containing the cellular proteins were blocked with blocking buffer (5% milk powder in TBS-T). Primary murine antibodies were used at a 1:1,000 (anti-human IL-16, clone 70719; R&D Systems), 1:3,000 (anti-human β-catenin, clone H102; Santa Cruz), and 1:5,000 dilution (anti-human GAPDH, clone 6C5; Santa Cruz), respectively. The secondary HRP-labeled antibody (R&D Systems) was used at a 1:5,000 dilution and signals were detected using chemiluminescent ECL solution.
8
Caspase-3 Activity Assay in NCKU-21 Treated Cells
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Seeded cells (106 cells/dish) were treated with 2 μM NCKU-21 for 0, 12, and 24 hr. After that, cells were trypsinized and lysed with chilled cell lysis buffer (#1067–100; BioVision, Milpitas, CA, USA). Cell lysates containing 100 μg of protein were diluted to a total volume of 50 μL and then mixed with 50 μL of 2× reaction buffer (#1068–20; BioVision). After that, 5 μL of DEVD-pNA (4 mM in DMSO; #1008; BioVision), a caspase-3 substrate, was added for incubation at 37°C for 2 hr. Caspase activity was determined by measuring the absorbance of the released pNA at 405 nm and is shown as the multiple of change compared to that of the control group (without NCKU-21 treatment).
9
Caspase-3 Activation Assay Protocol
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Cells were seeded in 100 mm plates, incubated for 24 h to permit adherence, and then treated with 0, 125 and 250 nM DS-3032b for 48 h. After cell lysis in Cell Lysis Buffer (BioVision), 50 μg protein were incubated with Ac-DEVD-AFC (Biomol) at 37°C for 1 h, and caspase 3 activity was measured with a multi-well fluorescence plate reader.
10
Protein Expression Analysis of Autophagy and Apoptosis Markers
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Total proteins were obtained using cell lysis buffer (BioVision, USA). ExKine Nuclear and Cytoplasmic Protein Extraction Kit (KTP3001, Abbkine, USA) was used to separate and extract proteins from the nucleus and cytoplasm, respectively. The protein samples were separated by 10% SDS-PAGE gels and subsequently transferred to nitrocellulose membranes. The primary antibodies including CKIP-1 (ab91489, abcam, USA), LC3 (ab51520, abcam, USA), Beclin-1 (ab207612, abcam, USA), p62 (ab109012, abcam, USA), KEAP1 (ab196346, abcam, USA), NRF2 (ab76026, abcam, USA), cleaved-caspase3 (ab2303, abcam, USA), caspase3 (ab13847, abcam, USA), cleaved-caspase7 (ab256469, abcam, USA), and caspase7 (ab32522, abcam, USA) are incubated with membrane at 4°C overnight. HRP-conjugated goat anti-rabbit IgG secondary antibody (ab6721, abcam, USA) was incubated at room temperature for 2 h. GAPDH (ab181602, abcam, USA) was presented as an internal control of total protein. Lamin B2 (ab8983, abcam, USA) was used as the internal control of nuclear protein. Enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc., USA) was used to visualize the protein bands in a BioRad ChemiDoc XRS Imaging system (Hercules, USA).
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