P. damselae cells at the logarithmic growth stage were diluted to 105–106 CFU/ml in PBS. The bacteria were pre-incubated with rCtl24 (50 μg/ml) for 2 h or not, and then with 5 μM of AlfB1 for 2 h at 28°C. After three washes, bacteria were stained using 1 μg/ml of propidium iodide (PI) (C0080, Solarbio, Beijing, China) and Hoechst 33258 (B8030, Solarbio) for 15 min at room temperature. After staining, the bacteria were washed three times and suspended in PBS. For flow cytometry, the cell suspension was examined using a flow cytometer (BD FACSAria Fusion, BD, Franklin Lakes, NJ, USA). The data were analyzed using the FlowJo software (FlowJo LLC, Ashland, OR, USA). For confocal imaging, the cell suspension was examined in the multi-track mode using a confocal microscope (LSM 900 with Airyscan, ZEISS, Oberkochen, Germany). The images were analyzed and presented using the ZEN software program (Zeiss).
C0080
Manufactured by Solarbio
Sourced in China
C0080 is a lab equipment product offered by Solarbio. It serves as a core function for laboratory applications, however a detailed description cannot be provided while maintaining an unbiased and factual approach. Additional information about the intended use or specific features of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Lsr 2, by BD (2 mentions)
Facsaria fusion, by BD (1 mentions)
Zen software program, by Zeiss (1 mentions)
Airyscan, by Zeiss (1 mentions)
Hoechst 33258, by Solarbio (1 mentions)
Multiskan fc, by Thermo Fisher Scientific (1 mentions)
C1359, by Merck Group (1 mentions)
L 929 cells, by Procell (1 mentions)
Ca1020, by Solarbio (1 mentions)
Dmi3000, by Leica (1 mentions)
Propidium iodide solution, by Solarbio (1 mentions)
Ckx41, by Olympus (1 mentions)
9 protocols using c0080
1
Bacterial Viability Assay with AlfB1
2
Cytotoxicity Evaluation of Biomaterials
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Cytotoxicity tests were conducted using the Live/Dead Cell Double Staining Kit and CCK-8 assay by exposing the samples to cultured L-929 cells (Procell Life Science & Technology Co., Ltd.). All sensors were sanitized with 75% alcohol and ultraviolet light, followed by bonding to the bottom of multiwall plates. L-929 cells were then cultured in the plates at 37°C under an atmosphere of 95% air and 5% CO2. For the Live/Dead Cell Double Staining Kit, cells were dyed by calcein-AM (C1359, Sigma-Aldrich Corp.) and PI (C0080, Solarbio Science & Technology Co., Ltd.) after culturing for 24, 48, and 72 hours, respectively. The fluorescent images were captured by a fluorescence microscope (BX51, Olympus, Corp.). For the CCK-8 assay, each plate was added with 10 μl of CCK-8 solution (CK04, Dojindo Molecular Technologies, Inc.) after culturing the cells for 24, 48, and 72 hours, respectively, followed by incubating for 2 hours. The cytotoxicity was obtained by measuring the absorbance at 450 nm using a microplate photometer (Multiskan FC, Thermo Fisher Scientific Inc.).
3
Cell Cycle Analysis by Flow Cytometry
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Cells inoculated into six-well plates were transfected using the same method as for the CCK-8 assay. After 48 h transfected, the cells were digested, collected, then stained with propidium iodide (C0080, solarbio, Beijing, China) for 30 min at room temperature. Finally, cell cycle classification and statistical analysis was carried out by flow cytometry.
4
Cell Cycle and Apoptosis Analysis
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The density of trypsinized cells was adjusted to 3 × 105 cells/mL, which were seeded in 6-well plates and cultured for 48 h. The single cell suspension was centrifuged at 1000 r.min−1 × 5 min followed by removal of the supernatant. Cells were fixed with 70% ice ethanol solution at − 20 °C or 1 h and then added with 20 μL RNA enzyme and reacted at 37 °C for 30 min. Subsequently, cells were incubated with 400 μL propidium iodide (PI) (C0080, Solarbio) for 15 min in the dark, on ice. The cell cycle was detected by a flow cytometer (BDLSR II, BD, FL, NJ, USA) with the excitation light wavelength at 488 nm. For detection of cell apoptosis, the cells were resuspended with 400 μL of Annexin V binding solution (CA1020, Solarbio) to 3 × 105 cells/mL and then incubated with 50 μL Annexin V-Fluorescein Isothiocyanate staining solution on ice, in the dark, for 15 min. After the addition of 10 μL PI staining solution, cell apoptosis was detected by flow cytometer also with the excitation light wavelength as 488 nm.
5
Apoptosis and Cell Cycle Analysis
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Apoptosis was assessed by annexin V/propidium iodide (PI) double staining. Y79 cells received treatment with 0.25% trypsin, and they were suspended and seeded into a culture plate at a density of 1 × 105 cells/mL, followed by three washes with PBS precooled at 4°C and trypsinization. After centrifugation at 290 g for 5 minutes, the supernatant was removed and the cells were resuspended in PBS again. The supernatant was discarded by centrifuging the 100-µL cell suspension (1 × 106 cells/mL) at 290 g for 5 minutes. Then, the cells were mixed with 500 µL 1× binding buffer, 5 µL FITC-labeled annexin V–FITC, and 10 µL PI in succession. The mixture underwent incubation with a flow cytometer (BDLSR II; BD Biosciences, San Jose, CA, USA) at room temperature for 5 to 10 minutes under dark conditions, followed by apoptosis analysis.
After centrifugation of the 100-µL cell suspension at 290 g for 5 minutes, the supernatant was discarded. The sample was washed twice with precooled 200 µL PBS, treated with 20 µL RNase for 30 minutes at 37°C, and cultured on ice for 15 minutes with 400 µL PI (C0080; Solarbio, Beijing, China) in the absence of light. Finally, cell cycle was examined by a flow cytometer (BDLSR II; BD Biosciences) at 488 mm.
After centrifugation of the 100-µL cell suspension at 290 g for 5 minutes, the supernatant was discarded. The sample was washed twice with precooled 200 µL PBS, treated with 20 µL RNase for 30 minutes at 37°C, and cultured on ice for 15 minutes with 400 µL PI (C0080; Solarbio, Beijing, China) in the absence of light. Finally, cell cycle was examined by a flow cytometer (BDLSR II; BD Biosciences) at 488 mm.
6
Cell Cycle Analysis by Flow Cytometry
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The treated GC cells were cultured for 48 h and then used the flow cytometry to analyze the cell cycle distribution. Briefly, the cells were fixed with 70% pre-cooled ethanol for 24 h. After being washed with cold PBS, 2 ×106 cells from each group were stained with propidium iodide (Solarbio, C0080) for 15 min. Then the stained cells were detected with flow cytometer. The cell cycle distribution was analyzed using the software FlowJo_v10.8.1.
7
Bacterial Membrane Permeability Assessment
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Cell membrane and cell wall permeability were assessed using propidium iodide (PI) and alkaline phosphatase (ALP) assay kits. The bacteria were cultured for 8 h to reach the mid-logarithmic phase. Subsequently, they were washed in 100 mM PBS (pH 7.3), and the cell density of the bacterial suspensions was adjusted to an OD600 of 0.4–0.5, ensuring uniform cell counts for each bacterial strain. Then, PI (Solarbio, C0080) was added to a final concentration of 10 mM and incubated at 28°C for 30 min. The fluorescence value was measured using a fluorescence plate reader (Thermo, Varioskan Flash) with excitation wavelength at 535 nm and an emission wavelength at 615 nm. The bacterial culture supernatants were collected and centrifuged at 12,000 r/min for 10 min. The ALP activity detection assay was performed using the AKP/ALP detection kit (Solarbio, BC2140) following the manufacturer's instructions. The measurements were performed in triplicate, and the data were presented as mean ± SE. Significance (p < 0.05) was determined using one-way ANOVA.
8
Plasmid Binding to Magnetic Beads
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Plasmids (80 μg, Hanbio Biotechnology Co., Ltd.) expressing the GFP or KLB gene were incubated in a 100-μl CMB suspension (0.5×109 MBs) for 15 min. The suspension was centrifuged at 400 × g for 5 min under 4°C to obtain plasmid-bound CMBs (10 (link)). The percentage of bound plasmid DNA and the payload mass of plasmid DNA in CMBs were calculated as follows: Percentage of bound plasmid DNA in CMBs=(DNAtotal-DNAfree)/DNAtotal ×100%; payload mass of plasmid DNA in CMBs=(DNAtotal-DNAfree)/CMB number (10 (link)). The plasmid DNA was stained by 10 μg/ml propidium iodide solution (C0080 Beijing Solarbio Science & Technology Co., Ltd.) for 10 min at room temperature. The combination of plasmid DNA and CMBs was validated using a fluorescence microscope (DMI3000, Leica Microsystems GmbH) and a flow cytometer (BD Biosciences).
9
Pyroptosis Evaluation by Propidium Iodide
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Propidium iodide (PI) staining was used to evaluate pyroptosis. For PI staining, the cells were washed with PBS 3 times, treated with 5 µM PI dye (C0080, Solarbio, Beijing, China), and then incubated at 37 °C in the dark for 20 min. After being removed from the PI dye, the cells were washed with PBS 2 times and observed under a fluorescence microscope (CKX41, Olympus, Tokyo, Japan). The percentage of PI positive cells was counted with Image J software (v1.8.0.112).
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