PMs were plated in 6-well plates or 24-well plates and pretreated with different inhibitors for 30 min at 37°C, including amiloride (1.5 mM, Selleckchem, USA), chlorpromazine hydrochloride (CPZ, 15 μg/mL, Selleckchem, USA), genistein (150 μM, Selleckchem, USA), and methyl-β-cyclodextrin (M-β-CD, 12mM, Sigma-Aldrich, Merck, Germany). amiloride, chlorpromazine hydrochloride (CPZ), genistein, and methyl-β-cyclodextrin (M-β-CD) were the inhibitors of micropinocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, and lipid raft-mediated endocytosis, respectively. Subsequently, PMs were transfected with CLP/siRNA (10:1, w/w) for 48 hours. The transfection efficiency was simultaneously detected by FCM and immunofluorescence (IF). For FCM, PMs were digested with Trypsin-EDTA solution (Servicebio, China) at 37°C for 10 min, and then cells were collected to detect the fluorescence intensity of FAM bound to siNLRP3. In addition, PMs cell membranes were stained with Dil (10 mg/mL, Beyotime, China), and nuclei were stained with Hoechst (1 mg/mL, Solarbio LIFE SCIENCES, China). Fluorescence intensity was detected by confocal microscopy (NIKON-A1R/STORM, NIKON, Japan).
Trypsin edta solution
Manufactured by Wuhan Servicebio Technology
Sourced in China
Trypsin-EDTA solution is a cell culture reagent used for the dissociation and detachment of adherent cells from cell culture surfaces. It contains the enzyme trypsin and the chelating agent EDTA, which work together to break down the proteins that attach cells to the substrate.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Amiloride, by Selleck Chemicals (1 mentions)
Genistein, by Selleck Chemicals (1 mentions)
A1r storm, by Nikon (1 mentions)
Methyl β cyclodextrin, by Merck Group (1 mentions)
Hoechst, by Solarbio (1 mentions)
Penicillin, by Wuhan Servicebio Technology (1 mentions)
Streptomycin, by Wuhan Servicebio Technology (1 mentions)
Mammary epithelial cell medium, by Procell (1 mentions)
Bt 549, by Thermo Fisher Scientific (1 mentions)
Mcf 10a, by Thermo Fisher Scientific (1 mentions)
Dmem medium with high glucose, by Thermo Fisher Scientific (1 mentions)
Mda mb 231, by Thermo Fisher Scientific (1 mentions)
Mcf 7, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Glucose, by Thermo Fisher Scientific (1 mentions)
3 protocols using trypsin edta solution
1
Inhibition of Endocytic Pathways for Nanoparticle Transfection
2
Cell Culture Protocols for Cancer and Normal Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human embryonic kidney cell line HEK293T, human breast cancer cell lines MCF7, MDA-MB-231, BT549, and SKBR3, and human normal breast epithelial cell line MCF10A were purchased from the Chinese Academy of Science, Shanghai (China). The MCF7, MDA-MB-231, and 293 T cells were cultured in DMEM medium with high glucose (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), while the BT549 and MCF10A cells were cultured in RPMI-1640 medium (Gibco, USA) and mammary epithelial cell medium (Procell, Wuhan, China), respectively. The growth media were added with 10% fetal bovine serum (Gibco, USA) and 100 U/mL penicillin and 100 μg/mL streptomycin (Servicebio, Wuhan, China). A 0.25% trypsin-EDTA solution (Servicebio, China) was also applied for cell passage cultivation. All cell lines were cultivated in a cell incubator at 37 °C and 5% CO2.
3
Cultivation of Human Kidney Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human ccRCC cell lines 786-O, A498, ACHIN, and Caki-2 and also human ordinary epithelial cell line HK-2 were supplied by the Chinese Academy of Sciences in Shanghai. Every one of the cells being cultured in DMEM media contained a lot of glucose (Gibco, USA). Penicillin (100 U/ml) and streptomycin (100 g/ml) had been mixed with 10% fetal bovine serum (PS; Servicebio, China) (FBS; Gibco, USA). 0.25% trypsin-EDTA solution (Servicebio, China) was employed for cell passage cultivation. The cells were cultured in a cell culture incubator at 37°C and 5% CO2.
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!