Anchorage-independent growth was assessed by plating 10,000 cells in 0.4% low melting temperature agarose (Seaplaque) in complete media on top of a 0.8% preformed agarose layer. Cells were grown for 9–14 days and colonies were stained with 0.5% crystal violet and destained with water. Microscopic images of the colonies were taken pre- and post-crystal violet staining. For growth curves, 250–1000 cells were plated on day 0 and grown for 5 days in culture. Four–five replicates for each cell line per day were assessed for cell viability by CTG assay. Cell viability results were normalized to luminescence at day 0. Three-dimensional culture was established by plating 250–500 cells from a single cell suspension onto a growth factor-reduced matrigel (Corning) layer, allowing cell migration into matrigel for 4–6 h. Cells were grown in complete media for 7–10 days prior to analysis. Three technical replicates were performed for each clone. Apoptosis was measured using the Guava Nexin Reagent per the manufacturer’s instructions and analyzed on a Guava flow cytometry system (Millipore).
Guava flow cytometry system
Manufactured by Merck Group
Sourced in United States
The Guava flow cytometry system is a compact and user-friendly instrument designed for automated cell analysis. It utilizes the principles of flow cytometry to provide cell-based measurements, including cell count, viability, and phenotyping. The system offers a streamlined workflow and intuitive software to facilitate efficient data acquisition and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 fitc apoptosis detection kit, by Merck Group (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Growth factor reduced matrigel, by Corning (1 mentions)
Annexin 5 fitc, by Southern Biotech (1 mentions)
Fetal bovine serum (fbs), by Avantor (1 mentions)
100 μm filter, by BD (1 mentions)
Quickextract, by Illumina (1 mentions)
Penicillin streptomycin, by Avantor (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Collagenase, by Worthington (1 mentions)
Dispase, by Roche (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit, by Merck Group (1 mentions)
9 protocols using guava flow cytometry system
1
Anchorage-independent Growth and 3D Culture Assessment
2
Apoptosis Evaluation of SKOV-3 Cells
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An Annexin V-FITC Apoptosis Detection Kit (Sigma, USA) was utilized to observe the apoptosis of SKOV-3 cells in response to C. majus. After 24 or 48 h of treatment, the cells were washed with PBS and exposed to Annexin V binding buffer, Annexin V-FITC conjugate (0.1 μg/ml) and PI (2 μg/ml). Following incubation at room temperature for 10 min protected from light, the fluorescence of Annexin V (530/30 nm filter) and PI (670 nm filter) in the samples were evaluated at 488 nm excitation with the Guava flow cytometry system (Millipore, USA).
3
Evaluating Chelidonine's Apoptotic Effects on Pancreatic Cancer
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To evaluate the apoptotic effects of chelidonine on human pancreatic cancer
cells, we performed Annexin-V/propidium iodide (PI) staining using the Annexin
V-FITC Apoptosis Detection Kit (Sigma, USA). The cells were treated with various
concentrations of chelidonine for 24 and 48 hours, detached using trypsin, and
washed twice with Phosphate-Buffered Saline (PBS). The cell suspension in PBS
was centrifuged at 200xg for 5 minutes, and the supernatant was
carefully removed by pipetting. The cell pellet was suspended in 500 µL
Annexin-V binding buffer, to which 0.1 µg/mL Annexin V-FITC conjugate and
2 µg/mL PI were added; the cells were incubated for 10 minutes at room
temperature with protection from light. The fluorescence of the samples was
detected using Guava flow cytometry system (Millipore, Massachusetts, USA) at an
excitation wavelength of 488 nm with a 530/30 nm band-pass filter to detect
Annexin V and 670 nm high-pass filter to detect PI.
cells, we performed Annexin-V/propidium iodide (PI) staining using the Annexin
V-FITC Apoptosis Detection Kit (Sigma, USA). The cells were treated with various
concentrations of chelidonine for 24 and 48 hours, detached using trypsin, and
washed twice with Phosphate-Buffered Saline (PBS). The cell suspension in PBS
was centrifuged at 200xg for 5 minutes, and the supernatant was
carefully removed by pipetting. The cell pellet was suspended in 500 µL
Annexin-V binding buffer, to which 0.1 µg/mL Annexin V-FITC conjugate and
2 µg/mL PI were added; the cells were incubated for 10 minutes at room
temperature with protection from light. The fluorescence of the samples was
detected using Guava flow cytometry system (Millipore, Massachusetts, USA) at an
excitation wavelength of 488 nm with a 530/30 nm band-pass filter to detect
Annexin V and 670 nm high-pass filter to detect PI.
4
Apoptosis Assay with Annexin V-FITC
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Floating cells were collected and adherent cells were trypsinized, filtered, and washed with ice-cold PBS. After centrifugation, 100,000 cells were resuspended in 100 μl binding buffer (0.01 M HEPES pH 7.4, 0.14 M NaCl, 2.5 mM CaCl2). Propidium iodide (Sigma Aldrich) and Annexin V-FITC (Southern Biotech) were added (1:100) and incubated at room temperature for 15 min. Four hundred microliters of binding buffer was added, and apoptosis analyses were performed using the Guava flow cytometry system (Millipore) using the same gate settings for each cell line. The experiment was performed with three biological replicates per condition, each with two technical replicates in three independent experiments.
5
Isolation and Characterization of Pancreatic Tumor Cells
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Pancreatic tumour cells from Pdx1-Cre-K-MADM-p53 mice were dissociated using a protease cocktail containing collagenase (Worthington), dispase (Roche) and trypsin-EDTA (Invitrogen) in Hank's balanced salt solution, quenched with fetal bovine serum (Hyclone) and passed through a 100 μm filter (Falcon) before plating in DMEM (CellGro) containing 10% fetal bovine serum and penicillin/streptomycin (VWR). Cells were grown in culture for 2–3 days and subject to FACS analysis using a Guava flow cytometry system (Millipore). Stable GFP+/tdTomato− (green) cell lines were obtained through serial passage and confirmed to be p53KO/KO by PCR genotyping of genomic DNA isolated using QuickExtract (Epicentre). For immunocytochemistry, primary cultured cells were grown on glass coverslips, fixed with PFA, permeabilized with 0.2% Triton X-100, blocked with 5% BSA in PBS and stained with rabbit anti-p53 primary antibody (Novacastra NCL-p53-CM5p, 1:200), donkey anti-rabbit Alexa 647 secondary antibody (Life Technologies A-31573, 1:500) and DAPI before mounting onto glass slides with Vectashield. Unlike with tissue sections, endogenous p53 was readily detectable by immunofluorescence on cultured cells.
6
Cell Cycle Analysis by Flow Cytometry
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To analyze the cell cycle profile, adherent cells were trypsinized, washed with PBS, and fixed with 70% ice-cold ethanol overnight. Cell pellets were resuspended in PBS containing 10–20 μg/ml RNase (Macherey-Nagel GmbH & Co. KG) and 20–40 μg/ml propidium iodide (Sigma Aldrich). Samples were incubated at room temperature in the dark for 30 min. Cell cycle profiles were obtained using the Guava flow cytometry system (Millipore) using the same gate settings for each cell line. Three biological replicates with each two technical replicates were processed in three independent experiments.
7
Apoptosis and Cell Viability Assays
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For analysis of apoptosis, cells were harvested, washed with PBS, and then stained with 7-AAD and Annexin V-PE. Samples were analyzed using a Guava flow cytometry system (Millipore). Cell viability is monitored with quantifying cellular proteins by the sulforhodamine B (SRB) assay. Cells were fixed with ice cold trichloroacetic acid (10% (wlv) and incubated for 60 min at 4°C. After washing with deionized water and air dried, SRB (Sigma) solution (0.4% w/v in 1% acetic acid) was added and incubated for 10 min at room temperature. After removing unbound SRB and washing with 1% acetic acid, cells were air dried. Bound protein stain was solubilized with of 10 mM Tris base (pH 10.5; 100 μ1), and OD565 nm was recorded using a microtiter plate reader.
8
Cell Cycle Analysis of Treated Cells
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To analyze the cell cycle profile under different treatment conditions, the cells were trypsinized and centrifuged to obtain pellets. To each cell pellet, 100% ethanol was added for fixation overnight. Following fixation, the cells were washed to allow for rehydration. Finally, for cell cycle analysis, propidium iodide was added and the profiles were obtained using the Guava flow cytometry system (Millipore). Three biological replicates were processed for each condition using the same gate settings.
9
Gossypol-induced Apoptosis Analysis
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Flow cytometry analysis was performed using the Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (Sigma-Aldrich). The cells were treated with various concentrations of gossypol for 24 and 48 h, detached using trypsin, and washed twice with PBS. The cell suspension in PBS was centrifuged at 200 ×g for 5 min, and the supernatant was carefully removed via pipetting. The cell pellet was suspended in 500 μl annexin V binding buffer, to which 0.1 μg/ml annexin V-FITC conjugate and 2 μg/ml PI were added, and the cells were then incubated for 10 min at room temperature in the dark. The fluorescence of the samples was detected using a Guava Flow Cytometry System (Millipore) at an excitation wavelength of 488 nm using a 530/30 nm band-pass filter to detect annexin V and a 670 nm high-pass filter to detect PI.
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