Facsflow solution

Manufactured by BD

Sourced in United States

FACSFlow solution is a sterile, isotonic diluent designed for use with flow cytometry instruments. It is formulated to provide the necessary conditions for optimal performance and stability of flow cytometry samples.

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FACSFlow (77 citations)

15 protocols using facsflow solution

Flow Cytometry Analysis of Fluorescent Samples

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Prior to flow cytometry analysis, 2 μl of fluorescent microspheres (1 × 10⁻³ of the stock Fluoresbrite YG microspheres; 0.75 μm; Polysciences) was added and the volume was adjusted to 500 μl by adding FACSFlow solution (10 mM phosphate-buffered saline, 150 mM NaCl, pH 7.4; Becton, Dickinson).
Samples were analyzed by using a BD FACSAria III flow cytometer (BD Biosciences, San Jose, CA). The cytometer was set up using an 85-μm nozzle and was calibrated daily using BD FACSDiva Cytometer Setup and Tracking (CS&T) software and CS&T beads (BD Biosciences). A 488-nm air-cooled argon-ion laser and the photomultipliers with 488/10-nm band pass filter for forward and side scatter (FSC and SSC) and with a 530/30-nm filter (with a 502 LP filter) was used for the detection of GFP, Sybr green, and MitoTracker. GelRed was excited with a yellow-green 561-nm laser and detected using a 610/20-nm filter with an LP 600-nm filter. CellMask DeepRed was excited with a 633-nm laser and detected with 660/20-nm filters. FM4-64 dye was excited with a 561-nm laser and detected at 780/60 nm with an LP 735-nm filter. FSC and SSC voltages of 300 and 350, respectively, and a threshold of 1,200 on FSC were applied to gate the bacteriophages and bacterial cell population.

Liu Y., Alexeeva S., Bachmann H., Guerra Martínez J.A., Yeremenko N., Abee T, & Smid E.J. (2022). Chronic Release of Tailless Phage Particles from Lactococcus lactis. Applied and Environmental Microbiology, 88(1), e01483-21.

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Quantifying CD47 Expression on RBCs

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CD47 expression on the membrane surface of RBCs was assessed using a monoclonal antibody against CD47, conjugated to allophycocyanin fluorochrome (eBioscience, San Diego, CA). For this purpose, an aliquot of washed and diluted peripheral blood containing 10 6 RBCs was incubated with CD47 antibody for 15 min in the dark. Subsequently, the sample was washed once with CellWash solution, resuspended in 250 mL of FACS Flow solution (Becton Dickinson) and 100,000 cells were acquired with the FACS Canto II flow cytometer (Becton Dickinson). The median fluorescence intensity served as the measure of expression level (relative molecule density), which was compared between the study group and the healthy control group.

Wiewiora M., Piecuch J., Sedek L., Mazur B, & Sosada K. (2017). The effects of obesity on CD47 expression in erythrocytes. Cytometry. Part B, Clinical cytometry, 92(6).

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CD14+ Monocyte Isolation and Analysis

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After isolation of CD14⁺ monocytes, 100 μl of the cell solution was mixed with 5 μl Human TruStain FcX™ (BioLegend, San Diego, USA) and incubated for 10 min to block unspecific Fc receptor bindings. Thereafter, we added 20 μl fluorescein isothiocyanate (FITC) anti-CD14 antibodies (clone: M5E2) (BD Biosciences, Heidelberg, Germany), followed by mixing and incubation for 15 min at RT in the dark. Then, the suspension was washed with 2 ml of FACS Flow solution (BD Biosciences, San Jose, USA) and centrifuged at 300 × g at RT for 5 min. The supernatant was removed and the pellet was resuspended in 300 μl FACSFlow. Gating was adjusted first on the cellular events in forward scatter (FSC) and side scatter (SSC), excluding particles smaller than 3 μm, followed by the assessment of CD14⁺ cells (Figure 1). Purity referred to all cellular events was calculated. All samples reached purities (fraction of CD14⁺ cells from all cells) of >80%.

Mayer D., Altvater M., Schenz J., Arif R., Karck M., Leuschner F., Weigand M.A., Uhle F, & Lichtenstern C. (2022). Monocyte Metabolism and Function in Patients Undergoing Cardiac Surgery. Frontiers in Cardiovascular Medicine, 9, 853967.

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Mitochondrial Membrane Potential Assay

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Fibroblasts were incubated with 40 nM Tetramethylrhodamine methyl ester perchlorate (TMRM; Sigma Aldrich) in HBSS-Ca/Mg solution (HEPES-buffered solution with 1.26 mM Calcium and 0.9 mM Magnesium) for 40 min at room temperature, protected from light. Labeling with the fluorescent probe MitoTracker Red CMXRos (250 nM, Life Technologies) was conducted for 30 min at 37 °C in medium protected from light. Fibroblasts were harvested with trypsin and washed twice with PBS. Cells were resuspended in FACS Flow solution (BD Biosciences, Franklin Lakes, NJ, USA) and cellular fluorescence intensity was measured using a four colours cytometer (FACSCalibur; BD Biosciences). For each sample, 10,000 events were recorded and the median of fluorescence intensity was used for the subsequent analyses with the CellQuest software (BD Biosciences).

Onesto E., Colombrita C., Gumina V., Borghi M.O., Dusi S., Doretti A., Fagiolari G., Invernizzi F., Moggio M., Tiranti V., Silani V, & Ratti A. (2016). Gene-specific mitochondria dysfunctions in human TARDBP and C9ORF72 fibroblasts. Acta Neuropathologica Communications, 4, 47.

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CellTrace™ Far Red Labeling Protocol

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Detached 2×10⁶ cells were washed and centrifuged in phosphate buffer solution 0.01 M pH 7.4 (PBS 1×). Cells were re-suspended with CellTrace™ Far Red (Invitrogen, Thermo Fisher Scientific) solution 1:1000 dilution in PBS 1X and incubated at 37°C bath Marie for 20 min, mixing gently every 5 min. Then, 4 ml of PBS 1X with 10% of FCS was added and incubated for 5 min at 37°C. All solutions were pre-heated at 37°C before use. After incubation, cells were centrifuged for 5 min at 1350×g, the supernatant discarded and cells re-suspended in sterile PBS 1× at a concentration of at least 1×10⁶ cells per ml. To ensure low residual volume in the cellular pellet, the cell suspension was transferred to 1.5 ml vials and centrifuged for 5 min at 3250×g. The supernatant was discarded, the vials were placed upside down for a minute and cells re-suspended for a final concentration of approximately 100 cells/nl. At least 10,000 cells were collected and re-suspended in FACs Flow solution (BD Biosciences, San Jose, CA, USA) for staining control, read at 660/20 nm in a FACsCanto II cytometer (BD Biosciences) and analyzed using FacsDiva 6.1 software (BD Biosciences).

Vargas-Patron L.A., Agudelo-Dueñas N., Madrid-Wolff J., Venegas J.A., González J.M., Forero-Shelton M, & Akle V. (2019). Xenotransplantation of Human glioblastoma in Zebrafish larvae: in vivo imaging and proliferation assessment. Biology Open, 8(5), bio043257.

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Isolation and Characterization of Human Mesenchymal Stem Cells

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Adult human MSCs from bone marrow were obtained from femoral biopsies of donors (age 50–78 years) undergoing total hip replacement, after signed informed consent and with approval of the local ethical committees (Erasmus MC number MEC-2004-142; Albert Schweizer Hospital number 2011.07). Cells from bone marrow aspirates were seeded at a density of approximately 50,000 nucleated cells cm⁻² in alpha-MEM (GIBCO), supplemented with 10% fetal calf serum (FCS), 1 ng ml⁻¹ FGF2 (AbD Serotec), 25 μg ml⁻¹ ascorbic acid-2-phosphate (Sigma-Aldrich), 1.5 μg ml⁻¹ fungizone, and 50 μg ml⁻¹ gentamicin. MSCs were isolated by their ability to adhere to plastic culture flasks. After 24 hr, nonadherent cells were washed out and adherent cells were cultured in standard conditions (5% CO₂ at 37°C) for 10 ± 2 (mean ± SD, n = 6 donors) days. Medium was renewed three times per week. When MSCs neared confluence, they were detached with 0.05% trypsin and characterized by flow cytometry. Cells were resuspended at 750,000 cells ml⁻¹ in FACSFlow solution (BD Biosciences).

Narcisi R., Cleary M.A., Brama P.A., Hoogduijn M.J., Tüysüz N., ten Berge D, & van Osch G.J. (2015). Long-Term Expansion, Enhanced Chondrogenic Potential, and Suppression of Endochondral Ossification of Adult Human MSCs via WNT Signaling Modulation. Stem Cell Reports, 4(3), 459-472.

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Multicolor Flow Cytometry Immunophenotyping

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Cells were resuspended at 750,000 cells ml⁻¹ in FACSFlow solution (BD Biosciences) and stained with antibodies against human CD45-PerCp (345809), CD73-PE (550257), CD140a-PE (556002), CD146-FITC (560846), CD166-PE (559263), CD271-Alexa Fluor (560326) (BD Biosciences), CD90-APC (FAB2067A), and CD105-FITC (FAB10971F) (R&D Systems), following the manufacturer’s guidelines. Unstained samples were used as negative controls. Flow cytometry analysis was performed using the BD FACSCanto II apparatus (BD Biosciences), and data were analyzed using BD FACSDiva software (BD Biosciences).

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Characterizing Breast Cancer Stem Cells

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Differentiation of CSCs was identified by the CD44⁺/CD24⁻ level. The cells were treated with DAC, trypsinized, and washed in PBS before being stained with monoclonal antibodies anti-CD44 and anti-CD24 (BD Biosciences, Franklin Lakes, NJ, USA). Tubes were incubated in the dark at room temperature for 30 minutes before FACSflow solution (BD Biosciences) was added. Using CellQuest Pro software (BD Biosciences), the CD44⁺CD24⁻ level was identified by quadrant analysis.
To analyze ABCG2 expression, BCSCs were fixed with FCM Fixation Buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 minutes and washed in PBS. Cold FCM Permeabilization Buffer (Santa Cruz Biotechnology) was added for 5 minutes at room temperature. Approximately 10⁵ cells were labeled with 2 µL ABCG2-FITC antibody (Santa Cruz Biotechnology) at 37°C for 30 minutes. Labeled cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences).
To analyze cell cycle, BCSCs were harvested by trypsinization, washed with PBS, and resuspended in 0.5 mL PBS. The tubes were gently vortexed, and 4.5 mL ice cold 70% ethanol was added dropwise over 30–60 seconds before incubation for 2 hours at 4°C. Cells were washed and stained with PI staining solution (Sigma-Aldrich) and analyzed using a FACSCalibur flow cytometer (BD Biosciences).

Phan N.L., Trinh N.V, & Pham P.V. (2015). Low concentrations of 5-aza-2′-deoxycytidine induce breast cancer stem cell differentiation by triggering tumor suppressor gene expression. OncoTargets and therapy, 9, 49-59.

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Single-Cell Flow Cytometry Analysis of Microbial Samples

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Cell concentrations and characteristics were determined via FC. To remove potential aggregates and allow single-cell analysis, 1 mL of the undiluted, unfiltered reactor samples were filtered with 20 µm syringe filters (Filcon, BD Biosciences, Belgium). Subsequently, 1000× dilutions were prepared in phosphate-buffered saline (PBS) in triplicates in 96-well plates, with dilution steps of 10-fold and a final volume of 200 µL. The technical replicates were stained with 1 v/v% SYBR Green I (SG) (100× concentrate in 0.22-µm-filtered dimethyl sulfoxide). After staining, the samples were incubated in the dark at 37 °C for 20 minutes (Props et al., 2016) (link).
After incubation, the samples were analysed for 60 seconds or until a total of 50 000 events were measured (ca. 50 µL) with a FACSVerse flow cytometer (BD Biosciences, Belgium) equipped with eight fluorescence detectors (527/32, 783/56, 586/42, 700/54, 660/10, 783/56, 528/45, and 488/45 nm), two scatter detectors, and a blue 20-mW 488-nm laser, a red 40-mW 640-nm laser, and a violet 40-mW 405-nm laser. The flow cytometer was operated with FACSFlow solution (BD Biosciences) as sheath fluid. Instrument performance was verified daily using FACSuite CS&T beads (BD Biosciences).

Sabbe K., D'Haen L., Boon N, & Ganigué R. (2023). Predicting the performance of chain elongating microbiomes through flow cytometric fingerprinting. Water research, 243.

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Erythropoiesis Evaluation in Sickle Cell Mice

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Erythropoiesis in bone marrow cells was assayed in HBB^A/A (controls), HBB^A/S (SCT), and HBB^S/S adult mice. Bone marrow cells suspensions were obtained, and isolated (1 × 10⁶ cells for a sample) were stained with anti-mouse Ter119 fluorescein isothiocyanate (FITC) and anti-mouse cluster of differentiation (CD)71 phycoerythrin (PE) antibodies (BD-Bioscience, San Jose, CA, USA) at a final concentration 1:100. Cells were incubated for 20 min at 4 C, washed with phosphate-buffered saline (5% bovine serum albumin), and re-suspended in fluorescence-activated cell sorting (FACS) flow solution (BD-Bioscience, San Jose, CA, USA). FACSCanto (BD-Bioscience, San Jose, CA, USA) flow cytometer was used to collect data and analyzed with FACSDiva software Version 6_1.3 (BD Biosciences, San Jose, CA, USA) and FlowJo V7_6.5. Erythropoiesis was estimated by the ratio between Ter119⁺/CD71⁺ and Ter119⁺/CD71⁻ in bone marrow cells from adult HBB^A/A, HBB^A/S, and HBB^S/S mice. Statistical differences between means were calculated with Student’s t-test (see Table 2).

Trucas M., Burattini S., Porcu S., Simbula M., Ristaldi M.S., Kowalik M.A., Serra M.P., Gobbi P., Battistelli M., Perra A, & Quartu M. (2023). Multi-Organ Morphological Findings in a Humanized Murine Model of Sickle Cell Trait. International Journal of Molecular Sciences, 24(13), 10452.

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