Total macrophage proteins were extracted using the Cell Lytic™ Cell Lysis Reagent (Sigma, St. Louis, MO, USA). ASM activity assay was performed according to a previously described procedure21 (link)). Briefly, 3 µl cell lysis was mixed with 3 µl ASM buffer [200 µm BODIPY®-labeled C12-Sphingomyelin (Thermofisher Scientific, Waltham, MA, USA)]. Then, it was diluted in an assay buffer [0.2 M sodium acetate (pH=5.0) containing 0.2 mM ZnCl2 and 0.2% Igepal CA-630]. Finally, it was incubated at 37 °C for 20 h. Furthermore, the reaction was terminated by adding ethanol. The hydrolytic product was detected and quantified using ultra performance liquid chromatography (UPLC) system (Acquity UPLC H-class, Waters, Milford, MA, USA). In this system, we used a reversed phase column (Acquity BEH Amide, 2.1 × 50 mm, 1.7 µm, Waters, USA).
Cell lytic cell lysis reagent
Manufactured by Merck Group
Sourced in United States
Cell Lytic™ Cell Lysis Reagent is a proprietary, non-ionic detergent-based reagent designed for the mild and efficient lysis of mammalian cells. It effectively disrupts cell membranes while preserving the integrity of cellular components, enabling the extraction of proteins and other biomolecules for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Acquity uplc h class, by Waters Corporation (1 mentions)
Bodipy labeled c12 sphingomyelin, by Thermo Fisher Scientific (1 mentions)
Acquity beh amide, by Waters Corporation (1 mentions)
Amersham hybond ecl, by GE Healthcare (1 mentions)
P38 αβ i, by Apexbio (1 mentions)
Msk1i, by Apexbio (1 mentions)
Pd l1, by Abcam (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
2 protocols using cell lytic cell lysis reagent
1
Quantification of Acid Sphingomyelinase Activity
2
Western Blot Analysis of Immune Pathway Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All of inhibitors (P38i, P38(αβ)i, MSK1i, MK2i) were obtained from ApexBio (USA). After LPS stimulation, neutrophils were lysed in ice-cold Cell Lytic cell lysis reagent (Sigma, USA) supplemented with Phosphatase Inhibitor Cocktail 2 (Calbiochem, USA) and EDTA-free protease inhibitor cocktail (Calbiochem, USA) for 5–10 min on ice. Cell lysates were scraped from 6-well plates, collected and centrifuged for 10 min at 8000g. Lysates were mixed 4:1 with 5× SDS-PAGE sample loading buffer and boiled for 8 min. Samples were subjected to SDS-PAGE on 8–12% gradient gels and transferred to Amersham Hybond-ECL (GE Healthcare, USA). Membranes were blocked for 1 h in 5% Milk in PBS-Tween (w/v), then incubated in 5% BSA in PBS-Tween (w/v) with antibodies against P38 (1:1000), P38α (1:1000), MSK1 (1:1000), MK2 (1:1000), phosphorylated p38 (1:1000), phosphorylated p38α (1:1000), phosphorylated MSK1 (1: 1000), phosphorylated MK2 (1:1000) (CST, USA), PD-L1 (1:1000) (Abcam, UK) and β-actin (1:1000) (Santa Cruz Biotechnology, USA), respectively. Then the membranes were incubated with secondary antibody conjugated to HRP (1: 2000–1:5000) (CST, USA). Protein bands were developed and detected using the Pierce ECL Western Blotting Substrate (Thermo Fisher, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!