A transwell assay was used to detect the invasion and migration of GC cells in the transfected and NC groups. After harvesting BGC-823 cells, the medium containing 10% serum was used to continue incubating the cells in the CO2 incubator (Forma Series II Water Jacket, Thermo) for 24 h at room temperature. The chamber was then removed. The cells inside the chamber were stained with crystal violet dye solution (C0121, Bi Yuntian) at room temperature for 30 min and gently rinsed with water severally. The chamber was subsequently removed, and the liquid in the upper chamber was absorbed. Next, the cells on the surface of the upper chamber membrane were carefully removed using a wet cotton swab; the membrane was carefully peeled off with a small forceps, and the bottom surface was dried. The cells were transferred to a glass slide (F518101, raw work), and the slide was sealed with neutral gum (E675007, aging). The three random fields were assessed under a microscope (Nikon), and the results were counted.
Forma series 2 water jacket
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Forma Series II Water Jacket is a laboratory incubator designed to maintain a controlled temperature environment for a variety of applications. The core function of this product is to provide a stable and uniform temperature-regulated chamber to support various experimental or analytical processes requiring a consistent thermal environment.
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Lab products found in correlation
4 protocols using forma series 2 water jacket
1
Transwell Assay for Invasion and Migration Analysis
2
Fluorescence-Based Cell Analysis Techniques
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Fluorescence signals were recorded by confocal laser scanning microscopy (CLSM, LSM 710, Carl Zeiss Microscopy GmbH, Göttingen, Germany). A FACS Calibur flow cytometer (BD Biosciences, San Jose, CA) was used to measure intracelluar reactive oxygen species and cell apoptosis. HEK-293 cells were incubated in a CO2 incubator (Thermo Scientific Forma Series II Water Jacket, Thermo Fisher Scientific, Inc., Waltham, MA). The fluorescence response of the biosensor cells was determined at different time points after exposure by high content screening (ImageXpress Micro XLS, Molecular Devices, USA).
3
Cardioprotective Effects of DATS-MION-PEG
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DATS@MION-PEG-LF (1 ~ 10 µg/mL) + GSH (2 mM) and saline of same volume (Control) were separately added to medium of cultured cardiomyocytes or neurons (n = 3). After incubation for 4 h, medium was removed and replaced with DMEM/F-12 without glucose and serum. Then cardiomyocytes/neurons were exposed to hypoxia (94 % N2, 5 % CO2, 1 % O2) for 4 h in a CO2 incubator (Forma SERIES II WATER JACKET, Thermo Scientific, MA, USA), followed by reoxygenation (5 % CO2) for 1 h. After which, the cell viability was evaluated by CCK-8 assay and compared with the Control.
After the hypoxia/reoxygenation procedure, lactate dehydrogenase (LDH) activities of each group were also measured to evaluate cytotoxicity using an assay kit (JianCheng, Nanjing, China) according to the manufacturer’s instructions. The absorbance was determined by a micro plate reader at 440 nm. The levels of cell apoptosis were also determined by flow cytometry by previous method [27 (link)].
After the hypoxia/reoxygenation procedure, lactate dehydrogenase (LDH) activities of each group were also measured to evaluate cytotoxicity using an assay kit (JianCheng, Nanjing, China) according to the manufacturer’s instructions. The absorbance was determined by a micro plate reader at 440 nm. The levels of cell apoptosis were also determined by flow cytometry by previous method [27 (link)].
4
Multiparametric Flow Cytometry of Side Population
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Vybrant DyeCycle Violet Ready Flow™ Reagent (Invitrogen) was added to cells in phenolfree DMEM:F12 medium and incubated at 37 o C in a 5% CO2 cell culture chamber (Forma Series II Water Jacket; ThermoFisher Scientific) for 30 minutes. The concentration of DCV was tested at both 1X and 2X, and based on these experiments (Fig S1), all future experiments used the concentration of 2X, or 160uL in 10 6 cells/ml.
For experiments involving ABC transporter inhibition, fumitremorgin C (FTC; Sigma) and (±) verapamil hydrochloride (VP; Sigma) were added to unwashed cells at final concentrations of 10uM and 50uM, respectively, after DCV incubation and kept in the same conditions for additional 30 minutes. Cell were then kept on ice in dark until sort, and 7-Amino-Actinomycin D (7AAD, 40 ug/ml, Sigma) was added to cell suspensions 10 minutes before analysis for dead cell discrimination. For experiments determining the identity of the side population, CD31 antibody conjugated to allophycocyanin (APC), BD Biosciences, BioLegend), was added to cells in DMEM:F12 at final concentration of 1:50 (24) and incubated on ice in the dark for 30 minutes before DCV incubation, which followed the same workflow as discussed above. Antibodies, dyes, and drugs used for all experiments are listed in Table 1.
For experiments involving ABC transporter inhibition, fumitremorgin C (FTC; Sigma) and (±) verapamil hydrochloride (VP; Sigma) were added to unwashed cells at final concentrations of 10uM and 50uM, respectively, after DCV incubation and kept in the same conditions for additional 30 minutes. Cell were then kept on ice in dark until sort, and 7-Amino-Actinomycin D (7AAD, 40 ug/ml, Sigma) was added to cell suspensions 10 minutes before analysis for dead cell discrimination. For experiments determining the identity of the side population, CD31 antibody conjugated to allophycocyanin (APC), BD Biosciences, BioLegend), was added to cells in DMEM:F12 at final concentration of 1:50 (24) and incubated on ice in the dark for 30 minutes before DCV incubation, which followed the same workflow as discussed above. Antibodies, dyes, and drugs used for all experiments are listed in Table 1.
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