Neutrophils (2 × 106, 200 μL) were incubated with the different inhibitors for 2 h in the same conditions used for the assays, and supernatants were collected for cell viability evaluation using the CytoTox® kit (Promega). Tween lysed neutrophils and purified lactate dehydrogenase were used as positive controls. Promastigotes (106) were incubated for 1 h with supernatants collected from neutrophils treated with the different inhibitors in the same conditions used for the assays. Then, PI (100 μg/ml; Sigma) was added immediately before reading on a FACSCalibur flow cytometer.
Facscalibur flow cytometer
Manufactured by Merck Group
Sourced in United States
The FACSCalibur flow cytometer is a laboratory instrument designed for the analysis of cells and particles. It utilizes the principles of flow cytometry to rapidly measure and analyze multiple physical and fluorescent characteristics of individual cells or particles as they pass through a laser beam. The FACSCalibur is capable of detecting and quantifying various parameters such as cell size, granularity, and the expression of specific cellular markers labeled with fluorescent dyes.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (3 mentions)
Annexin 5 fitc, by BD (2 mentions)
Cytotox kit, by Promega (1 mentions)
Azd6244, by Selleck Chemicals (1 mentions)
Cellquest software, by BD (1 mentions)
Rnase a, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Annexin 5 fluoroisothiocyanate apoptosis detection kit, by Merck Group (1 mentions)
Facs lysing solution, by BD (1 mentions)
Trypan blue staining, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Primary antibody against cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Fitc conjugated secondary antibody, by Merck Group (1 mentions)
Rnaase a, by Roche (1 mentions)
Wst 1 assay, by Roche (1 mentions)
Annexin 5 fitc cell apoptosis detection kit, by Beyotime (1 mentions)
Alexa fluor 488 azide, by Thermo Fisher Scientific (1 mentions)
Guava easycyte, by Merck Group (1 mentions)
Guava easycyte mini flow cytometer, by Merck Group (1 mentions)
Facs caliber, by Beckman Coulter (1 mentions)
15 protocols using facscalibur flow cytometer
1
Neutrophil-Promastigote Interaction Assay
2
Evaluating Apoptosis Induction in Colon Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human colon carcinoma cell lines were treated, in duplicate, with equimolar concentrations (20 μM) of each agent that inhibits SMO (GDC-0449; JS Research), MEK (AZD6244; Selleckchem) or GLI (GANT61; Calbiochem). Following 72 hr exposure, cells were collected by trypsinization and incubated with Annexin V FITC (BD Biosciences, CA) and propidium iodide (Sigma, MO) prior to analysis using a FACSCalibur flow cytometer. Raw data were analyzed by CellQuest software [36 (link)-39 (link)].
3
Cell Cycle Analysis by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SCs were plated at a density of 3 × 106 cells/well in a six-well plate and incubated for 48 h. After incubation, cells were sequentially trypsinized, washed with PBS, fixed overnight in 70% ice-cold ethanol (Sigma-Aldrich Co., St. Louis, MO, USA), at 4°C, resuspended in PBS plus 30 µg/ml RNase-A (Sigma-Aldrich Co., St. Louis, MO, USA), and incubated at 4°C for 5 min. Following the addition of FACS buffer (PBS + 2% FBS, Euroclone, Milan, Italy) and PI (1 mg/ml; Sigma-Aldrich Co., St. Louis, MO, USA), cells were incubated at 4°C for 30 min and finally DNA content was measured using a FACSCalibur flow cytometer. CellQuest software (Becton Dickinson, CA, USA) was used to quantify the distribution of cells in each cell cycle phase: sub-G1 (dead cells), G0/G1, S, and G2/M. Twenty thousand live events were collected for each sample and experiments were performed in triplicate. Apoptotic cells with hypodiploid DNA content were measured by quantifying the sub-G1 peak in the cell cycle pattern (43 (link), 44 (link)).
4
Apoptosis detection via flow cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apoptosis was evaluated with an AnnexinV-fluoroisothiocyanate apoptosis detection kit according to the instructions of the manufacturer (Sigma-Aldrich, Shanghai) and analyzed with use of a FACSCalibur flow cytometer and CellQuest software as previously described [20 (link)-22 (link)].
5
Annexin V FITC Apoptosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HT29 and SW480 cells were treated, in duplicate, as described in the Figure legends. At the end of treatment, cells were collected by trypsinization and incubated with Annexin V FITC (BD Biosciences) and propidium iodide (Sigma) prior to analysis using a FACSCalibur flow cytometer, as described [21 (link), 23 ]. Data were analyzed using FlowJo software.
6
Quantifying Phagosomal OVA Degradation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytometric analysis of OVA-degradation in the phagosome has been described in detail elsewhere [24 (link)]. Briefly, beads were prepared as above with 100% OVA coating. DCs were pulsed with the bead-bound OVA for 30 min at 18°C, washed in ice cold PBS. Fetal calf serum (FCS) flotation was performed 3 times by overlaying the cells on 3 mL of fetal calf serum and centrifuging at 900rpm to remove non-internalised beads. Cells were chased at 37°C in CO2-independent medium for the 30, 60, 90 or 120 min, resuspended in homogenization buffer (8% sucrose in PBS, 3 mM imidazole, 1 mM dithiothreitol) and disrupted mechanically with 2 mL syringes and 22-gauge needles (Terumo Medical). After centrifugation, the post-nuclear supernatant was transferred to 96-well conical microplates followed by labelling on ice with a rabbit polyclonal antibody against OVA (Sigma) and an Alexa647-labelled secondary antibody and analysed using a FACSCalibur flow cytometer.
7
Immunophenotyping of Blood Samples by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunophenotyping of blood samples by flow cytometry and lymphocyte subset identification was performed according to Table S1 . In brief, 25 μL of EDTA blood was mixed with 1 μL of each antibody and incubated in the dark at room temperature for 15 minutes. About 500 μL of 1× FACS lysing solution (Becton Dickinson) was added to each tube and incubated in the dark for 10 minutes to lyse erythrocytes. Cells were washed twice with PBS and fixed with 1% formaldehyde (Sigma)/PBS and data acquired on a FACSCalibur Flow Cytometer. CellQuest was used for analysis using the gating strategy in Fig. S5 .
8
Evaluating 786-O Cell Viability and Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Trypan blue staining (Invitrogen) was used to measure cell viability. Five hundred healthy 786-O SFCs and MACs or sorted cells were cultured with RPMI-1640 medium containing 10% FBS and the viability of the cells was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) method (Sigma). The cell growth curves were drawn accordingly. For cell cycle analysis, SFCs and MACs were harvested and fixed with 70% ethanol for 24 h. Cells were washed with PBS, stained with 40 μg/mL propidium iodide and 1 μg/mL RNase (Sigma), and then analyzed on a FACSCalibur flow cytometer. The percentage of proliferating cells was calculated using the formula: (G2/M + S)/(G0/G1 + S + G2/M) × 100%.
9
Apoptosis Detection by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells treated with etoposide ± SM-164 were trypsinized, washed by centrifugation with PBS, fixed with 4% paraformaldehyde for 15 min, washed with PBS and permeabilized with ice-cold 90% methanol for 30 min on ice. Washed samples were then incubated with primary antibody against cleaved caspase-3 (Cell Signaling, Danvers, MA, USA) for 2 h at room temperature. After three washes in PBS, cells were subjected to FITC-conjugated secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h. A total of 10,000 cells for each sample was analyzed by FACSCalibur flow cytometer. Data were analyzed using Flowing software.
10
Cell Proliferation and Cell Cycle Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Relative cell proliferation was determined with the WST-1 assay (Roche, Mannheim, Germany) in 96-well culture plates as described previously. 40 For cell cycle analysis, cells were fixed in 70% ice-cold ethanol and were treated by RNAase A (50 mg/ml; Roche). Cells were stained in PBS/2 mM EDTA/ 0.1% Triton-X-100 containing 20 mg/ml propidium iodide (Sigma, Deisenhofen, Germany) 30 min before analysis on FACS Calibur flow cytometer. 40
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!