Colony formation assay is used to demonstrate the colony forming capacity of a single cell during treatment.T238 cells were grown in 6-well culture plates (1000 cells/well) and after 24 h, they were treated with different concentration of metformin, vemurafenib and the combination of both. Treated cells were incubated at 37 °C and 5% CO 2 for 10-14 days till the formation of optimal clones. After incubation, cells were xed with methanol and stained with Crystal violet. Annexin V-FITC assay Annexin V-FITC assay is used to detect early apoptosis where the cells were stained by both PI and Annexin V-FITC. Expression of phosphatidylserine, which occurs through the binding of Annexin V-FITC to the cancer line cell surface, indicates apoptotic cells. Further staining with propidium iodide, a nonpermeable DNA dye, indicates necrotic cells. After 48 hours of treatment with metformin, vemurafenib or the combination of both, T238 cells were detached by trypsin, washed with PBS and then resuspended with Annexin V binding buffer. To each aliquot of 96 µl cell suspension, 1 µl of Annexin V-FITC conjugate and 12.5 µl of propidium Iodide (PI) solution were added and incubated for 10 min on ice in the dark. This was diluted to a nal volume containing 250 µl/assay with ice-cold 1X Annexin V binding buffer and analyzed using a BD FACSVERSE ow cytometer with an in-built BD FACSuite TM Software.
Facsverse ow cytometer
Manufactured by BD
Sourced in United States
The FACSVerse is a flow cytometer designed for immunophenotyping and cell analysis. It is capable of simultaneous multi-parameter detection and measurement of various cellular characteristics, such as size, granularity, and expression of specific markers on the cell surface or within the cell. The FACSVerse provides reliable and reproducible data for research and clinical applications.
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Lab products found in correlation
Facsuite software, by BD (3 mentions)
Facsuite cs t research beads, by BD (1 mentions)
Flow sensor, by BD (1 mentions)
Flow cytometry sub micron particle size reference kit, by Thermo Fisher Scientific (1 mentions)
Pspax2, by Addgene (1 mentions)
Anti phospho histone h2ax ser139, by BioLegend (1 mentions)
Pvsv g, by Addgene (1 mentions)
Facsdiva software, by BD (1 mentions)
Tryple express 1x, by Thermo Fisher Scientific (1 mentions)
Facsaria 3 ow cytometer, by BD (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Ebioscience foxp3 transcription factor staining buffer kit, by Thermo Fisher Scientific (1 mentions)
Cytometric bead array, by BD (1 mentions)
Fcap array v3, by BD (1 mentions)
Flow jo software version 7, by Tree Star (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Jc 1 dye, by Thermo Fisher Scientific (1 mentions)
Pas staining kit, by Abcam (1 mentions)
Indocyanine green icg, by Merck Group (1 mentions)
13 protocols using facsverse ow cytometer
1
Evaluating Cancer Cell Viability and Apoptosis
2
Flow Cytometry Particle Size Analysis
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All samples were analyzed using a BD FACSVerse ow cytometer with a three-laser con guration (488 nm, 640 nm, and 405 nm) equipped with a BD Flow sensor using the manufacturer settings. BD FACSuite CS&T research beads were used daily to calibrate and perform quality control checks per the manufacturer's instructions. All samples were analyzed in 5ml polystyrene round bottom tubes using a medium sample ow rate (60ul/min) with a normal sheath core stream uid velocity (5.5 m/s). Data was collected using BD FACSuite software and exported to BD Flowjo software for analysis. Event counts and volume metrics were exported to Microsoft Excel for nal calculations. For submicron analysis, Forward scatter (FSC) and side scatter (SSC) voltage was adjusted to 765 and 465, respectively, with a FSC threshold of 10,000. Settings were evaluated using Invitrogen's Flow Cytometry Size Kit Calibration Kit containing non uorescent polystyrene microspheres ranging in size from 1-15um and Invitrogen's Flow Cytometry Submicron Particle Size Reference Kit containing green-uorescent polystyrene microspheres ranging in size from 0.02-2um. Quality control checks for the size gate was selected using Invitrogen's 1um non uorescent polystyrene microspheres (Fig. 1).
3
MTH1 Knockdown and DNA Damage Analysis
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Cells received treatment for three days were harvested and xed in 75% ice cold ethanol and kept at -20 ºC overnight. Cells were spun down and washed with ice-cold PBS containing 2% FBS for three times. The cell suspension was incubated with an Anti-phospho-histone H2AX (Ser139) (BioLegend Cat. No. 613404, CA, USA) antibody for 1 h at room temperature. After a brief wash, 10,000 stained cells were collected and analyzed by a BD FACS Verse ow cytometer. shRNA construction and MTH1 knockdown MTH1 shRNA sequences were designed by an online tool from Sigma-Aldrich website or obtain from literature. Two MTH1 targeting sequences: shRNA#1 5'-GAAATTCCACGGGTACTTC-3' and shRNA#2 5'-CGACGACAGCTACTGGTTT-3' were synthesized and subcloned into a pLKO.1-TRC vector via Age1/EcoRI restriction sites. For transfection, the above plasmids, empty vector together with the packaging plasmids pVSVg (#8454, Addgene) and psPAX2 (#12260, Addgene) were transfected into HEK293T cells at approximately 60% con uence using polyethylenimine (PEI) for 48 h. The culture medium of U251 cells was replaced with the above lentivirus-containing supernatants with the addition of 1:1000 of polybrene and was centrifuged for 2 hrs at 37 ºC for viral infection. After puromycin selection for 3-7 days, the infected cells were collected for validation of MTH1 knockdown.
4
Analyzing Platelet-Cell Interactions by Flow Cytometry
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Flow cytometry experiments were performed using 24-wells plates and 500μl of platelets suspension. Experiments were run in triplicates and collected at different time points. After co-culture, platelets were harvested from cell media after centrifugation at 105 x g for 15 min to eliminate cell fragments. Cells were washed twice with PBS 1X to remove any remaining platelets and cell colonies were dissociated with Tryple Express 1X (Life Technologies). Cells and platelets suspensions were xed with 3.7% paraformaldehyde (PFA) (Electron Microscopy Sciences) for 20 min at room temperature.
For the analysis of cell activation, platelets were incubated with mouse anti-human PAC- Between primary and secondary antibody incubations, cells and platelets were washed with FACS Buffer (PBS 1X, 5% FBS, EDTA 2Mm) and PBS-EDTA 2mM, respectively.
Both cells and platelets were analyzed in the BD FACSVerse™ ow cytometer equipped with three lasers: violet (405nm), blue (488nm) and red (633nm) (BD Bioscience) using BD FACSuite™ software (BD Bioscience) for acquisition or by FACS ARIA III™ ow cytometer equipped with four lasers: violet (405nm), blue (488nm), yellow/green (531nm) and red (633nm) (BD Bioscience) using BD FACSDiva™ software (BD Bioscience) for acquisition and FlowJo™ for analysis (FlowJo, LLC-BD Bioscience). Flow cytometry gating strategy is described in Fig. S2.
For the analysis of cell activation, platelets were incubated with mouse anti-human PAC- Between primary and secondary antibody incubations, cells and platelets were washed with FACS Buffer (PBS 1X, 5% FBS, EDTA 2Mm) and PBS-EDTA 2mM, respectively.
Both cells and platelets were analyzed in the BD FACSVerse™ ow cytometer equipped with three lasers: violet (405nm), blue (488nm) and red (633nm) (BD Bioscience) using BD FACSuite™ software (BD Bioscience) for acquisition or by FACS ARIA III™ ow cytometer equipped with four lasers: violet (405nm), blue (488nm), yellow/green (531nm) and red (633nm) (BD Bioscience) using BD FACSDiva™ software (BD Bioscience) for acquisition and FlowJo™ for analysis (FlowJo, LLC-BD Bioscience). Flow cytometry gating strategy is described in Fig. S2.
5
Splenocyte Isolation and Cytokine Analysis
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Spleen were removed and mechanically dissociated into single cell suspensions. Splenocytes were incubated with Fc block (CD16/32) for 10 minutes to block non-speci c binding before stained with the conjugated antibodies. For surface staining, cells were incubated with speci c antibodies for 30 minutes on 4℃ followed by washing twice. For intracellular cytokine staining, cells were stimulated with phorbol myristate acetate and ionomycin and in the presence of monensin (eBioscience) for 5 h prior to staining with the BD Fixation/Permeabilization Solution kit. Treg cells were xed and permeabilized using the eBioscience Foxp3/transcription factor staining buffer kit (Invitrogen) according to manufacturer's instruction. Flow cytometry data were acquired on a BD FACSVerse ow cytometer (BD Bioscience) and analyzed using FlowJo version 10 software.
6
Cell Cycle Analysis by Flow Cytometry
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The cells were trypsinized, xed in 70% ice-cold ethanol, and incubated with propidium iodide and RNase A for 30 min at room temperature. The cell cycle was detected using the FACSVerse ow cytometer (BD, San Jose, CA, USA).
7
Quantifying Inflammatory Cytokines in Alcoholic Liver Cirrhosis
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The serum concentrations of in ammatory cytokines interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70 and TNFα were measured using human in ammation kit (Cytometric Bead Array; BD Biosciences, San Jose, CA, USA) by FACS Verse™ ow cytometer according to the manufacturer's instructions. Cytokine measurements were performed using cryopreserved serum. Individual cytokine concentrations were computed using FCAP Array™ v3.0 Software (BD Biosciences). The minimum and maximum detection limits were 10 and 2,500 pg/mL, respectively. Serum samples obtained from 23 healthy donors and 24 patients with alcoholic liver cirrhosis (LC) with Child-Pugh Grade A or B were also evaluated as controls. Characteristics of the patients with Child-Pugh Grade A or B are shown in supplementary table 1.
8
Macrophage Differentiation Phenotypes
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To determine the macrophage differentiation phenotypes, BMDMs were isolated from C57BL/6 mice and NET and/or ET models were established as previously reported. Then, adherent cells were collected for ow cytometry analysis of F4/80 and CD86. RAW264.7 cells were separately stained with a monoclonal antibody speci c for PE-CD86. Both cell cultures were then analyzed using a FACSVerse ow cytometer (BD Biosciences, San Jose, CA, USA).
9
Annexin V-FITC Apoptosis Assay
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The annexin V-uorescein isothiocyanate (FITC) apoptosis assay kit (ApexBio, K2003, China) was used to determine whether cell death was induced by apoptosis according to the literature (Yunxia et al. 2013) (link).Brie y, the cells were treated with different drugs and collected, resuspended in 500 µl of Annexin V binding buffer, incubated with Annexin V-FITC and Propidium iodide for 15 min at room temperature in the dark, then analyzed by FACSverse ow cytometer (BD, USA). The results were shown as a dot plot graph, with the X-axis corresponding to the relative PI staining and the Y-axis corresponding to the log of the FITC signal.
10
Detecting TLR7 in Human Basophils
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To detect expression of TLR7 in human blood basophil cells, blood cells were challenged with or without ASWE,HDME or PPAE (all at a concentration of 1.0 μg/ml) for 60 min at 37℃, respectively, and 2 μg/ml brefeldin A was also added into the tube.Cells were then incubated with human Fc receptor blocking solution and a live/dead cell dye (Zombie Green TM Fixable Viability kit for 15 min, and each labelled monoclonal antibody including APC/Cy7-conjugated mouse anti-human CCR3, PE/Cy7-conjugated mouse anti-human CD123, PerCP/Cy5.5-conjugated mouse anti-human HLA-DR was added into the tube.
After red blood cells being lysed, resuspended leucocytes were xed and permeabilized using Cyto x/Cytoperm TM Fixation/Permeabilization kit according to the manufacturer's instructions.This was followed by adding a PE-conjugated anti-human TLR7 antibody into the tube and incubated at 4℃ for 30 min.
Finally, cells were resuspended in uorescence activated cell sorting (FACS)-ow solution and analysed with FACS Verse ow cytometer (BD Biosciences, San Jose, CA). A total of 10,000 events in live cell gate were analysed for each sample. Data were analysed with FlowJo software version 7.0 (Treestar, Ashland, OR, USA). Dead cells and doublets were excluded from analysis by live/dead cell dyes.
After red blood cells being lysed, resuspended leucocytes were xed and permeabilized using Cyto x/Cytoperm TM Fixation/Permeabilization kit according to the manufacturer's instructions.This was followed by adding a PE-conjugated anti-human TLR7 antibody into the tube and incubated at 4℃ for 30 min.
Finally, cells were resuspended in uorescence activated cell sorting (FACS)-ow solution and analysed with FACS Verse ow cytometer (BD Biosciences, San Jose, CA). A total of 10,000 events in live cell gate were analysed for each sample. Data were analysed with FlowJo software version 7.0 (Treestar, Ashland, OR, USA). Dead cells and doublets were excluded from analysis by live/dead cell dyes.
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