SA-49 was synthesized as described previously and dissolved in DMSO [21 (link)]. LY294002, Go6976, 5Z-7-Oxozeaenol and Torin1 were purchased from Selleck (Beijing, China). Cycloheximide (CHX), MG132, and Bafilomycin (Baf) were purchased from Sigma (St. Louis, MO, USA). Antibodies against PD-L1, TFEB, MITF, H3, PKCα, p-GSK3β (Ser9), cleaved caspase 9 and 3 were purchased from Cell Signaling (Danvers, MA, USA). Anti-GSK3β and GAPDH antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Anti-PD-L1-PE, IgG-PE and FoxP3 antibodies were purchased from eBioscience (San Diego, CA, USA). Antibodies against p-PKCα (T638), CD3 and Ki67 were obtained from Abcam (Cambridge, MA, USA). The probes LysoTracker and DAPI were purchased from Invitrogen (Carlsbad, CA, USA). Human PD-1 Fc recombinant protein and IL-2 were purchased from R&D Systems (Minneapolis, MN, USA).
Bafilomycin baf
Manufactured by Merck Group
Sourced in United States
Bafilomycin (Baf) is a macrolide compound that functions as a selective inhibitor of vacuolar-type H+-ATPase (V-ATPase). It is commonly used as a laboratory tool to study the role of V-ATPase in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human pd 1 fc protein, by R&D Systems (2 mentions)
Anti gsk3β, by Santa Cruz Biotechnology (1 mentions)
Torin1, by Selleck Chemicals (1 mentions)
Gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Anti pd l1 pe, by Thermo Fisher Scientific (1 mentions)
Igg pe, by Thermo Fisher Scientific (1 mentions)
Go6976, by Selleck Chemicals (1 mentions)
Foxp3 antibody, by Thermo Fisher Scientific (1 mentions)
Ly294002, by Selleck Chemicals (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Chloroquine cq, by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Phrodotm red dye conjugates, by Thermo Fisher Scientific (1 mentions)
M 2585, by Bachem (1 mentions)
Fluoxetine, by Merck Group (1 mentions)
Celltiter 96 non radioactive cell proliferation assay, by Promega (1 mentions)
4 protocols using bafilomycin baf
1
Investigating PD-L1 Regulation and Signaling
2
PD-1/PD-L1 Axis Regulation in TCM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies are listed in Supporting Information Table S1 . Traditional Chinese medicine (TCM) chemical monomers were purchased from Shanghai Standard Technology Co., Ltd. (Shanghai, China). CHX, MG132, bafilomycin (Baf), chloroquine (CQ), and Hoechst 33342 were purchased from Sigma (St. Louis, MO, USA). Human PD-1 Fc recombinant protein was purchased from R&D Systems (Minneapolis, MN, USA). The plasmids pcDNA3-HA-Ub (18712) obtained from Addgene (Watertown, MA, USA), pCMV3-PD-L1-Myc (HG10084-CM) and pCMV3-CSN5-Flag (HG17128-CF) plasmids were purchased from Sino Biological Inc. (Beijing, China).
3
Phagocytosis Assay with Zymosan and Amyloid-β
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The phagocytosis assay was performed with fluorescent zymosan bioparticles from Saccharomyces cerevisiae (pH-sensitive pHrodoTM Red dye conjugates; Life Technologies, Carlsbad, CA, USA) or oligomerized FITC–amyloid-β (M-2585; BACHEM, Bubendorf, Switzerland). Primary MGCs were seeded at a density of 2 × 104 cells/well in 96-well plates and cultured for 48 h. The cells were treated with LPS (1 μg/mL) and/or fluoxetine (5 µM) and/or bafilomycin (Baf; 10 nM; Sigma–Aldrich) for 24 h in serum-free DMEM. The cells were treated with zymosan red or FITC–amyloid-β in serum-free media for 2–3 h. In some experiments, we conducted time-lapse imaging using a fluorescence microscope (DP80; Olympus, Japan). Blindly labeled images were analyzed to quantify phagocytosis.
4
Modulating Cellular Processes with Selective Inhibitors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteasome inhibitors MG132 (Calbiochem), bortezomib (Bort) (Santa Cruz Biotechnology), and lactacystin (Lact) (Santa Cruz Biotechnology) were dissolved in dimethyl sulfoxide (DMSO) and used at a concentration of 1, 20, and 0.5 μM, respectively. Endosomal acidification inhibitor bafilomycin (Baf) (Sigma Aldrich), dynamin-inhibitor dynasore (Dyn), and inhibitor of Na + H + exchanger EIPA were dissolved in DMSO and used at 0,2, 80, and 50 μM, respectively. PI3K inhibitors wortmanin (Wort) and LY294002 (LY294) and E1 inhibitor Pyr-41 were dissolved in DMSO and used at 10 μM. DNA synthesis inhibitor AraC and protein synthesis inhibitor cycloheximide (CHX), were dissolved in double-distilled H2O (ddH2O) and used at 100 μg/ml. Working solutions at the indicated concentrations were freshly prepared in DMEM supplemented with 2% FBS. Before infection, cells were pretreated with these drugs for 1 h except for Pyr-41 where cells were pre-treated for 8 h. Drugs were present throughout the course of the experiment unless otherwise indicated. To determine the aforementioned working concentrations, we analyzed cell viability and cytotoxicity tests for each drug treatment with CellTiter 96 Non-radioactive Cell Proliferation Assay (Promega) and following the manufacturer’s instructions. Based on these results, we selected the optimal non-toxic concentrations for setting up all the experiments.
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!