MKN-45 and MKN-28 cells were plated separately in a glass-bottom cell culture dish (NEST) with serum-free media overnight. The next day, cells were fixed with 4% paraformaldehyde for 20 min at room temperature. After that, cells were washed with PBS and blocked with 1% bovine serum albumin (BSA, Fcmacs Biotech, Nanjing, China) for 30 min at room temperature; then, the cells were incubated with a final concentration of 1 μM of peptide FITC-CM 7, FITC-CM 14, and FITC (Solarbio, Shanghai, china) alone for 1 h at room temperature away from the light. Following that, cells were washed with PBS twice and stained with 8 μM of DiI for 10 min at room temperature. Subsequently, cells were washed with PBS twice and stained with Hoechst 33342 (Wanleibio, Shenyang, China) for 20 min at room temperature. Finally, cells were washed with PBS three times and visualized under a confocal microscope (LSM 800, Zeiss, Germany) with an oil-immersion objective lens (×63, Plan-Apochromatlan, Zeiss, Germany).
Hoechst 33342
Manufactured by Wanlei
Sourced in China
Hoechst 33342 is a fluorescent dye used for the detection and quantification of DNA in biological samples. It binds to the minor groove of double-stranded DNA, emitting a blue fluorescent signal that can be detected using appropriate imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Varioskan flash plate reader, by Thermo Fisher Scientific (2 mentions)
Sytox green, by Thermo Fisher Scientific (2 mentions)
Plan apochromatlan, by Zeiss (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Glass bottom cell culture dish, by NEST Biotechnology (1 mentions)
Fluorescein isothiocyanate (fitc), by Solarbio (1 mentions)
Cyclin b1, by Wanlei (1 mentions)
Cleaved caspase 8, by Wanlei (1 mentions)
Cyclin e, by Wanlei (1 mentions)
Nur77, by Santa Cruz Biotechnology (1 mentions)
Cleaved parp, by Santa Cruz Biotechnology (1 mentions)
C jun, by Santa Cruz Biotechnology (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3, by Wanlei (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Tlrl 3pelps, by Thermo Fisher Scientific (1 mentions)
Histone primary antibody, by ABclonal (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg secondary antibody, by Abcam (1 mentions)
Laser co focus light microscopy, by Nikon (1 mentions)
6 protocols using hoechst 33342
1
Peptide Binding Assay in Cell Lines
2
Apoptosis Regulation by CTD Compound
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CTD (purity ≥98%, product number: MB6525) was purchased from Dalian Meilun Biotechnology Co., Ltd. and added with an appropriate amount of DMSO solution to dissolve into a 32 mM stock solution, stored at −20°C for further use. The antibodies against caspase 3, cleaved caspase 3, cleaved caspase 8, Cyclin E, Cyclin B1, CDK2, p21, p27, and p53 were obtained from Wanlei Biotechnology (Shenyang, China). The antibodies against Nur77, PARP, cleaved PARP, c-Jun, Jun-B, Bcl-2, and Bax were purchased from Santa Cruz Biotechnology (CA, USA). The antibodies against β-actin, mouse, or rabbit IgG were obtained from Sigma-Aldrich (St. Louis, MO, USA). Hoechst 33342 was obtained from Wanlei Biotechnology (Shenyang, China). Other chemicals were obtained from Sigma-Aldrich, unless indicated otherwise.
3
Hoechst 33342 Staining for Cell Nuclei
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment at 12-well plates for 48 h, cells were washed with PBS for 3 times. Then, Hoechst 33342 (Wanlei Biotechnology, Shengyang, China) dissolving in PBS was added into each well. The plates were kept at room temperature for 10 min and avoided from light. Finally, the plates were washed with PBS again and images were captured on a Zeiss fluorescence microscope (Carl Zeiss, Germany).
4
Hoechst Staining for Apoptosis Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured in 24-well plates that were plated with glass slides in advance and treated with SA followed by H/R as previously described. After that cells were fixed with 4% paraformaldehyde for 30 min, and then incubated with 0.5 ml Hoechst 33342 solution (Wanlei, Shenyang, China) for 5 min at room temperature. After that cells were washed twice with PBS and the changes in nuclear morphology were observed under fluorescence microscopy with 350 nm excitation and 461 nm emission. The number of Hoechst-positive nuclei per optical field (at least 5 fields) was counted in three independent experiments and further calculated the ratio of apoptotic cell.
5
Macrophage Extracellular Traps Induced by S. pluranimalium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The matured BMDMs were plated onto poly L-lysine-coated coverslips, and incubated with LPS (500 ng/mL, 4 h, Invitrogen, #tlrl-3pelps) before being stimulated with S. pluranimalium strain STP1 (MOI = 25, 100 min). The macrophage extracellular traps (METs) were determined using anti-histone (1:100, ABclonal, #A2348), anti-MPO (1:100, Biorbyt, #orb16003), SYTOX Orange`1 (5 mM, Invitrogen, #S-11368), Hoechst 33,342 (2 mM, Wanleibio, #WLA042a), and Alexa Fluor®488-conjugated anti-rabbit IgG antibodies. Images were acquired and analysed using a Laser Co-focus light microscope (Nikon). For scanning electron microscopy (SEM) analysis, the samples were sputtered with gold and then measured by SEM according to the manufacturer´s instructions (Servicebio Technology Co., LTD, Wuhan, China). To quantify METs formation, the extracellular DNA content in the supernatants was determined using SYTOX Green (5 mM, Invitrogen, #S7020) using a Varioskan Flash plate reader (Thermo Scientific). Cells treated with zymosan (1 mg/ml) served as positive controls.
6
Visualization and Quantification of Neutrophil Extracellular Traps in C. perfringens Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The matured BMDMs were seeded on 12-mm 0.01% poly-l-lysine–coated coverslips in 24 well-plates and then were incubated with LPS for 4 h before being infected with C. perfringens strain ATCC13124, CP1 or CP3 at a multiplicity of infection (MOI = 20) for 90 min, respectively. Cells were counterstained with Sytox Orange (5 μM, Invitrogen, #S-11368) and Hoechst 33342 (2 μM, Wanleibio, #WLA042a), and then were incubated with MPO primary antibody (Biorbyt, #orb16003), ELANE Polyclonal antibody (ABclonal, #A13015) or histone primary antibody (ABclonal, #A2348) and Alexa Fluor®488-conjugated anti-rabbit IgG secondary antibody (Abcam, #ab150077). Extracellular traps were visualized on a Laser Co-focus light microscopy (Nikon) and images were analyzed (Schindelin et al., 2012 (link)). To quantify extracellular traps formation, extracellular DNA content in the supernatants were determined. LPS primed-BMDMs were stimulated with bacteria (MOI = 20), Sytox Green (5 μM, Invitrogen, #S7020) was added after 90 min. The cell supernatants were collected for fluorescence assay using a Varioskan Flash plate reader (Thermo Scientific).
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!