One milliliter of cell culture (OD595 of 0.55–1.2) was pelleted (4,000 g, 2 min, 4°C) and resuspended in 1 ml 70% ice‐cold ethanol. Fixed cells were kept at 4°C if cells from multiple time points were collected. Five hundred microliter fixed cells were centrifuged (3,000 g, 5 min, 25°C) and washed twice with 1 ml 50 mM sodium citrate. Cell pellets were resuspended by vortexing. After the last washing step, pellets were treated with 500 μl 50 mM sodium citrate +0.1 mg/ml RNase A for at least 1.5 h at 37°C. Subsequently, 500 μl 50 mM sodium citrate +2 μM Sytox Green (Thermo Fisher Scientific) was added and samples were mixed by vortexing. To break apart cell aggregates, samples were sonicated for 5 s on a 550 Sonic Dismembrator (Thermo Fisher Scientific) before being transferred to 5 ml FACS tubes (Becton Dickinson). Flow cytometry measurements were performed on a BD LSRII SORP Analyzer (Becton Dickinson) using a 488 nm excitation laser (flow rate = low; mode = linear). Per sample 20,000 cells were counted, and the data were postprocessed and visualized with FlowJo.
Facs tube
Manufactured by BD
Sourced in United States, United Kingdom, Germany
FACS tubes are specialized laboratory containers used for flow cytometry analysis. They are designed to hold and transport samples for processing in a flow cytometer instrument. The tubes provide a standardized format to ensure consistent sample handling and data collection during the cytometric analysis process.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (11 mentions)
Facscanto 2, by BD (6 mentions)
Lsrii flow cytometer, by BD (5 mentions)
Propidium iodide, by Merck Group (4 mentions)
Facscalibur instrument, by BD (4 mentions)
Cellquest, by BD (4 mentions)
Facsdiva software, by BD (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Facs lysing solution, by BD (3 mentions)
Anti cd45 fitc, by Thermo Fisher Scientific (3 mentions)
Anti cd14 fitc, by BD (3 mentions)
Anti cd44 pe, by BioLegend (3 mentions)
Anti cd90 pe, by Thermo Fisher Scientific (3 mentions)
Anti cd29 pe, by BioLegend (3 mentions)
Canine fc receptor binding inhibitor, by Thermo Fisher Scientific (3 mentions)
Anti cd34 pe, by R&D Systems (3 mentions)
Facscelesta, by BD (3 mentions)
Sytox green, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sytox blue viability dye, by Thermo Fisher Scientific (2 mentions)
102 protocols using facs tube
1
Cell Cycle Analysis by Flow Cytometry
2
Annexin V Assay for Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Exposed phosphatidyl serine was measured using an Annexin V FITC Assay kit (Cayman, Ann Arbor, MI, USA) following the manufacturer’s indications. Briefly, 2.5 million NEU/mL were placed into FACS tubes (FALCON; Becton Dickinson, Franklin Lakes, NJ, USA) in the presence of 40 µL of APTP, HHC, or HLT sera or 10 µL of zymosan (50 µg/mL) for 8 hours. A total of 200 µL of binding buffer was then added, and the tubes were centrifuged to remove the supernatants. The cell pellets were resuspended in 50 µL of staining solution (FITC-Annexin V-propidium iodide) for 10 minutes. Next, the cells were analyzed using a Facscalibur flow cytometer (Becton Dickinson) and the data were processed with the FlowJo software for early apoptosis and late apoptosis.
3
Meiotic Cell Cycle Analysis by FACS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two hundred microliters of meiotic cells (at 2 OD/ml) were collected at each time point, resuspended in 70% ethanol and stored overnight at 4°C. Samples were then spun down, resuspended in 200 μl of solution A (50 mM sodium citrate, 0.1 mg/ml RNAse A (Sigma, Cat# R6513), 0.2 mg/ml Proteinase K (Thermo Scientific™, Cat# EO0491)) and incubated 2 h at 50°C. Cells were pelleted again and resuspended in 500 μl of 50 mM sodium citrate containing 2 μM of SYTOX Green (Invitrogen™, Cat# S7020) and transferred to 5 ml FACS tubes (Becton Dickinson, Cat# 352008). After vortexing, samples were analyzed on the LSR II (BD Biosciences) instrument and BD FACS Diva software. Single cells were gated based on forward and side scattering, FITC-A, and FITC-W. Ten thousand events were counted per sample. Data were analyzed using FlowJo 10 software.
4
3D Microcarrier-based Cell Invasion Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Approximately 500 Cytodex Microcarrier beads (Sigma Aldrich, St. Louis, MO, USA, C3275) per 1.25 × 104 cells/ml were mixed in FACS tubes (Becton, Dickinson and Company, Allschwill, Switzerland, Falcon T7597-5J) and incubated at 37 °C for 6 h with a mild shaking of the tubes at every one hour. Cells, which were not adhered to the beads, were removed by washing the beads with fresh medium. Cell-coated microbeads were re-suspended in 2.5% bovine collagen 1 (Advanced BioMatrix, San Diego, CA, USA, 5005-B) and seeded in μ-clear 96 well plate (Greiner CELLSTAR®, 655090). Following the polymerization of collagen, the cell coated microbeads were overlaid with fresh medium and treated with appropriate concentrations of growth factors/cytokines or with HGF or with c-Met inhibitors. After 24 h, cells were fixed with 4% paraformaldehyde (PFA) and stained with Hoechst. Images were acquired using an ImageXpress Micro 2 automated microscope (Molecular Devices, LLC, USA) as described for the zone infiltration assay. Cell invasion is expressed as the average of the distance invaded by the cells from the circumference of the bead as measured by our cell dissemination counter software aMDIcs.
5
Monitoring T Cell Activation and Antigen Presentation
6
Cell Cycle Analysis by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 48 hours post transfection, the cells were briefly centrifuged and the media was aspirated. Cells were stained with 300 µl of propidium iodide solution containing 0.1 mg/ml propidium iodide (Sigma), 3 µl/ml Triton-X 100 (Sigma), 1 mg/ml sodium citrate (Sigma) and 20 µg/ml RNase (Sigma) for 30 minutes at 4 °C. The stained cells were immediately transferred to BD FACS tubes and acquired in BD FACS Canto.
The data obtained was analyzed using FlowJo software. DNA histograms were analyzed to quantitate sub G0, G0/G1, S and G2/M populations. Minor shifts were observed in the G0/G1 and G2/M populations and hence manual gating was done to analyze these populations.
The data obtained was analyzed using FlowJo software. DNA histograms were analyzed to quantitate sub G0, G0/G1, S and G2/M populations. Minor shifts were observed in the G0/G1 and G2/M populations and hence manual gating was done to analyze these populations.
7
Platelet Activation and Surface Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FACS analysis was performed as reported by us and others previously23 (link),48 . Briefly, platelets were isolated, dissolved in tyrode buffer and 50 µl diluted cells were incubated in FACS buffer (2 mM EDTA, 0.5% FCS, in PBS) in BD FACS tubes with 5 µl of the following antibodies: LeoF2 Emfret M025-1 FITC labeled (GPIIbIIIa), XiaB4 Emfret M051-1 FITC labeled (GPIX), Xia X2 Emfret M043-1 FITC labeled (GPIba) and JON/A Emfret M023-2 (GPIIbIIIa activated, PE labeled). The respective IgG controls served as a control. After incubation for 30 min at 4 °C, 1 ml of FACs buffer was added, vortexed, and the cells centrifuged at 300 g for 10 min, then the pellet resuspended in FACS flow and immediately analyzed in a FACScan. In order to stimulate platelets with thrombin, isolated platelets were incubated with 1 mM CaCl2 and 500 µg thrombin (from bovine plasma, Merck 12374) for 30 min in FACS buffer with the respective antibodies and then processed and analyzed the same way.
8
Apoptosis Detection by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Analysis of the PI-positive apoptotic cells was performed by flow cytometry. A549 cells were treated with 10 nM PT-1 for 30 min with or without irradiation with the 405 nm laser for 1 h. The cells were further incubated for 12 h and harvested. Then the cells were washed two times with ice-cold PBS. The cells were placed (at up to 106/ml) in BD FACS tubes and incubated with 5 mg/ml PI (Sigma-Aldrich) in PBS with 10% FBS for 30 min at room temperature. The cells were then washed in ice-cold PBS. The PI fluorescence was measured in the FL-1 channel of a FACSAria instrument (BD, San Jose, California, USA).
9
Apoptosis Quantification by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected by trypsinization with 0.05% Trypsin-EDTA and washed twice with cold PBS. To include the floating cells, prior to trypsinization, culture medium was collected and floaters were spun down by centrifugation in FACS tubes (BD Falcon, CA, USA) at 4°C for 5 min. The cells were then stained with annexin V-FITC and PI according to the manufacturer’s instructions (BD Pharmingen, CA, USA) on ice. Cells were then analyzed by flow cytometry (BD FACSCanto II) within 1 h.
10
α-Synuclein Treatment of Pericytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pericytes were cultured in 24 well plates (Nunc) before treatment with α-syn or vehicle (PBS). Following timed treatments, cultures were incubated with Trypsin-1 mM EDTA (Gibco) at 37 °C for 5 min to harvest cells and placed in FACS tubes (Falcon). Cells in suspension were incubated in 8% PFA for 10 min and then centrifuged at 300×g for 5 min. Subsequently cells were washed twice with PBS-T for permeabilization for 10 min each. Cells were incubated with primary antibodies at 1:500, including a no-primary control overnight at 4 °C. The cells were pelleted, and the primary antibody removed and cells were washed before addition of secondary antibody including the nuclear stain 7-Amino-Actinomcyin D (7-AAD) (BD Pharmingen; 559925, 1:100) for 3 h at room temperature. Samples were then run on Accuri C6 flow cytometer (BD Biosciences) until 5000 cellular events had been recorded (Supplementary Fig. 10 ).
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!