3MA was purchased from MilliporeSigma. RPMI-1640 medium, fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) were purchased from Gibco; Thermo Fisher Scientific, Inc. The Hoechst 33342/PI kit and Baf-A1 were purchased from Beijing Solarbio Science & Technology Co., Ltd. The Cell Counting Kit (CCK)-8 kit, Ad-GFP-LC3B and actin mouse monoclonal antibody (cat. no. AF0003) were purchased from the Beyotime Institute of Biotechnology. The Annexin V-FITC apoptosis detection kit was purchased from BD Biosciences. The pro-caspase-3 (cat. no. ab32150), cleaved caspase-3 (cat. no. ab32042), caspase-8 (cat. no. ab32397), caspase-9 (cat. no. ab32068), PARP (cat. no, ab32138) and cleaved PARP (cat. no. ab32561) antibodies were purchased from Abcam. LC3B (cat. no. 2775S), Beclin-1 (cat. no. 3738S), AMPK (cat. no. 2532S), phosphorylated (p-)AMPK at Ser 485 (cat. no. 4184S), Akt (cat. no. 9272S), p-Akt at Ser473 (cat. no. 9271S), mTOR (cat. no. 2972S), p-mTOR at Ser2448 (cat. no. 2971S), p70S6K (cat. no. 9202S) and p-p70S6K at Thr389 (cat. no. 9205S) antibodies were purchased from Cell Signaling Technology, Inc.
Hoechst 33342 pi kit
Manufactured by Solarbio
Sourced in China
The Hoechst 33342/PI kit is a fluorescent staining solution used in cell biology applications. It contains Hoechst 33342, a cell-permeable dye that binds to DNA, and propidium iodide (PI), a dye that can only enter cells with compromised membranes. This kit allows for the simultaneous staining and detection of live and dead cells.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Baf a1, by Solarbio (1 mentions)
Evos m5000, by Thermo Fisher Scientific (1 mentions)
Facscanto flow cytometry, by BD (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Cell counting kit 8 (cck8), by Beyotime (1 mentions)
Binding buffer, by Keygen Biotech (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Deae cellulose de52, by Cytiva (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Keygen Biotech (1 mentions)
Whatman, by Cytiva (1 mentions)
8 protocols using hoechst 33342 pi kit
1
Investigating Autophagy and Apoptosis Mechanisms
2
Apoptosis and Necrosis Assay for SH-SY5Y Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SH-SY5Y cell apoptosis and necrosis were detected by using an ANNEXIN V/Dead Cell.
Apoptosis kit (194785, Thermo Fisher Scientific, USA) and a Hoechst 33342/PI kit (CA1120, Solarbio, Beijing, China) according to the operation protocol [38 , 39 ]. Briefly, after incubation and treatment, SH-SY5Y cells were harvested using 0.05% trypsin, centrifuged to remove the medium, washed twice with ice-cold PBS, and resuspended in 500 μL of 1X binding buffer. The 5 × 104 cells (30 μL of cell suspension) were mixed and incubated with 5 μL of Annexin V-FITc reagent at 37°C for 30 min under dark conditions, and followed by propidium iodide (PI, 5 μL) staining for 5 min. Stained cells were measured via a FACSCanto flow cytometry (BD, Biosciences, USA). The percentage of cells in quadrant 2 (Q2) and 4 (Q4) were quantitatively analyzed, generating the total percentage of apoptotic cells (apoptotic index) both early and late apoptotic stage. All experiments were performed in triplicate.
Meanwhile, after incubation and treatment, cells were washed three times with FBS-free DMEM and then incubated with the 10 μg/mL Hoechst 33342 staining solution for 20 min at room temperature in the dark, followed by PI staining for 5 min. Finally, after washed two times with DMEM, the cells were examined using a fluorescence microscope (EVOS M5000, Thermo Fisher Scientific, USA).
Apoptosis kit (194785, Thermo Fisher Scientific, USA) and a Hoechst 33342/PI kit (CA1120, Solarbio, Beijing, China) according to the operation protocol [38 , 39 ]. Briefly, after incubation and treatment, SH-SY5Y cells were harvested using 0.05% trypsin, centrifuged to remove the medium, washed twice with ice-cold PBS, and resuspended in 500 μL of 1X binding buffer. The 5 × 104 cells (30 μL of cell suspension) were mixed and incubated with 5 μL of Annexin V-FITc reagent at 37°C for 30 min under dark conditions, and followed by propidium iodide (PI, 5 μL) staining for 5 min. Stained cells were measured via a FACSCanto flow cytometry (BD, Biosciences, USA). The percentage of cells in quadrant 2 (Q2) and 4 (Q4) were quantitatively analyzed, generating the total percentage of apoptotic cells (apoptotic index) both early and late apoptotic stage. All experiments were performed in triplicate.
Meanwhile, after incubation and treatment, cells were washed three times with FBS-free DMEM and then incubated with the 10 μg/mL Hoechst 33342 staining solution for 20 min at room temperature in the dark, followed by PI staining for 5 min. Finally, after washed two times with DMEM, the cells were examined using a fluorescence microscope (EVOS M5000, Thermo Fisher Scientific, USA).
3
Cell Viability and Apoptosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell Counting Kit-8 was used to measure the cell viability according to the manufacturer’s protocol (Beyotime, China). SHSY-5Y cells were seeded in the 96-well plates at a density of 5 × 103 cells/well. After the incubation, the culture medium was replaced by basal medium containing 10% CCK-8 solution, and the incubation continued for another 1 h at 37°C. Absorbance was measured at 450 nm by a microplate reader (Bio-Rad, United States).
Flow cytometry assay was performed to measure the cell apoptosis. Cells were suspended in 100 μl of binding buffer (Nanjing KeyGen Biotech. Co., Ltd., China). Then, apoptosis was detected using Hoechst 33342/PI Kit according to the manufacturer’s protocol (Solarbio, Beijing, China). Cells were stained with 5 μl of Hoechst 33342 solution and incubated in the dark at room temperature for 10 min. Then, 5 μl of propidium iodide (PI) was added to the cell suspension. Hoechst 33342/PI-stained cells were analyzed immediately by using Scalibur Flow Cytometer (BD Biosciences, United States).
Flow cytometry assay was performed to measure the cell apoptosis. Cells were suspended in 100 μl of binding buffer (Nanjing KeyGen Biotech. Co., Ltd., China). Then, apoptosis was detected using Hoechst 33342/PI Kit according to the manufacturer’s protocol (Solarbio, Beijing, China). Cells were stained with 5 μl of Hoechst 33342 solution and incubated in the dark at room temperature for 10 min. Then, 5 μl of propidium iodide (PI) was added to the cell suspension. Hoechst 33342/PI-stained cells were analyzed immediately by using Scalibur Flow Cytometer (BD Biosciences, United States).
4
Dual Fluorescent Staining of Aortic Slices
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The slices of the aorta were double stained with a Hoechst 33342/PI kit (Solarbio, CA1120) according to the manufacturer’s instructions. Briefly, dewaxed slices were double-stained with Hoechst 33342 and PI at 4°C for 40 min. Subsequently, the stained slices were observed with a fluorescence microscope (Olympus, BX50).
5
Dexamethasone-Induced Apoptosis in hBMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells at passage 3 were grown in 24-well plates and were treated with 10−6 mol/L Dex for 10 days. From day 1 to day 10, a chromatin dye Hoechst 33342/PI Kit (Solarbio, Beijing, China) was used to assess the morphology of the apoptotic cells, following the manufacturer’s instructions. Apoptotic cells, which showed morphological characteristics, such as chromatic agglutination, karyopyknosis, and nuclear fragmentation, were identified and counted under a fluorescent microscope. Necrotic cells emitted a red hyperfluorescence and a blue hyperfluorescence. In addition, Hoechst 33342/PI staining assay of the hBMSCs treated with various concentrations of Dex (10−8 mol/L, 10−7 mol/L, and 10−6 mol/L) was also performed for 7 days. The hBMSCs were treated with the solvent of Dex as the control. The experiment was performed in triplicate.
6
Dexamethasone-induced Apoptosis in hBMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells at passage 3 were grown in 24-well plates and were treated with 10 -6 mol/L Dex for 10 days. From day 1 to day 10, a chromatin dye Hoechst 33342/PI Kit (Solarbio, Beijing, China) was used to assess the morphology of the apoptotic cells, following the manufacturer's instructions. Apoptotic cells, which showed morphological characteristics, such as chromatic agglutination, karyopyknosis, and nuclear fragmentation, were identi ed and counted under a uorescent microscope. Necrotic cells emitted a red hyper uorescence and a blue hyper uorescence. In addition, Hoechst 33342/PI staining assay of the hBMSCs treated with various concentrations of Dex (10 -8 mol/L, 10 -7 mol/L, and 10 -6 mol/L) was also performed for 7 days. The hBMSCs were treated with the solvent of Dex as the control. The experiment was performed in triplicate.
7
C2C12 Cell Morphology Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The C 2 C 12 cell morphology was measured using the Hoechst 33342/PI kit (Solarbio, China). The method used was as described by Chen et al. [24] with a slight and incubated with 1 mL of cell staining buffer, 5 μL of Hoechst 33342 (excitation 360 nm/emission 465 nm) buffer and 5 μL of PI staining (excitation 488 nm/emission 620 nm) buffer for 30 min at 4 °C in the dark. The percentage of survival and apoptotic cells were then analyzed using CLSM (Leica, Germany).
8
Protein Extraction and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DEAE Cellulose DE-52 was purchased from Whatman (Maid-stone, Kent, UK).
The Hoechst 33342/PI kit (Solarbio, China) and radio immunoprecipitation assay lysis buffer were purchased from Keygen (Solarbio, China). The BCA protein assay kit was purchased from Thermo Fisher (Pittsburgh, PA). Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Abcam (Abcam, Cambridge, Mass., USA). All other chemicals and reagents were purchased from Sigma (Saint-Louis, MO, USA).
The Hoechst 33342/PI kit (Solarbio, China) and radio immunoprecipitation assay lysis buffer were purchased from Keygen (Solarbio, China). The BCA protein assay kit was purchased from Thermo Fisher (Pittsburgh, PA). Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Abcam (Abcam, Cambridge, Mass., USA). All other chemicals and reagents were purchased from Sigma (Saint-Louis, MO, 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!