Flow buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States

Flow buffer is a specialized liquid solution designed to facilitate the proper operation of flow cytometry instruments. Its primary function is to create a stable and consistent flow of cells or particles through the measurement area of the cytometer, ensuring accurate and reliable data acquisition.

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Lab products found in correlation

Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions) Triton x 100, by Merck Group (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Permeabilization buffer, by Thermo Fisher Scientific (1 mentions) Cd16 cd32, by BD (1 mentions) Clostridium histolyticum, by Merck Group (1 mentions) Cell strainer 70 m nylon, by Corning (1 mentions) Zombie uv, by BioLegend (1 mentions) Ultracomp ebeads compensation beads, by Thermo Fisher Scientific (1 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions) Rbc lysis buffer, by Merck Group (1 mentions) Mouse igg2a, by Thermo Fisher Scientific (1 mentions) Cellrox green reagent, by Thermo Fisher Scientific (1 mentions) Flow buffer, by BD (1 mentions) C6 plus flow cytometer, by BD (1 mentions) Pe conjugated anti human cd19 antibody, by Thermo Fisher Scientific (1 mentions)

4 protocols using flow buffer

Multiparameter Flow Cytometry Analysis

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Samples were fixed with 2% PFA for 20 min at RT, then wash with flow buffer (eBioscience). Samples were then incubated with PE-conjugated anti-human CD19 antibody (eBioscience) or isotype controls for 20 min at RT, then wash with flow buffer, followed by incubation with p62-Alexa Fluor 488 antibody for 60 min at RT. Samples were then washed with flow buffer, and analyzed with BD C6 plus flow cytometer.
For intracellular ROS measurement, 1X10⁶ cells in 500 µl medium per well were seeded in 24-well plates, and cultured overnight. 1 µl CellROX Green Reagent (Invitrogen) was added to each well and incubated for 30 min. Cells were then washed 3 times with PBS, and fixed with 2% PFA for 20min at RT, followed by extensive washes and then incubated with PE-conjugated anti-human CD19 antibody (eBioscience) for 20min at RT, before subjected to flow cytometry.

Wang L., Howell M.E., Sparks-Wallace A., Hawkins C., Nicksic C.A., Kohne C., Hall K.H., Moorman J.P., Yao Z.Q, & Ning S. (2019). p62-mediated Selective autophagy endows virus-transformed cells with insusceptibility to DNA damage under oxidative stress. PLoS Pathogens, 15(4), e1007541.

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Flow Cytometry Analysis of Differentiated Cells

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After singularization with ACCUTASE_™, cells were fixed in 1% paraformaldehyde for 20 minutes. Primary antibodies were diluted in flow buffer (5% wt/vol BSA (ThermoFisher) in PBS containing 0.1% Triton X-100 (Sigma)) according to Supplementary Table 1 and added to fixed cells overnight at 4 °C. Secondary antibodies (1:1000 dilution in flow buffer) were incubated for 30 minutes at room temperature prior to analysis. Samples were run on a BD FACSCalibur flow cytometer. H9 hESCs were used to make CPCs and CMs and 19-9-11 and H1 hPSCs were used to make ECs for flow cytometry analysis. Statistical analysis was performed using one-way ANOVA with Tukey’s HSD post hoc analysis, Student’s t-test, Mann-Whitney test, or the Kruskal-Wallis test with Dunn’s post hoc analysis.

Dunn K.K., Reichardt I.M., Simmons A.D., Jin G., Floy M.E., Hoon K.M, & Palecek S.P. (2019). Coculture of Endothelial Cells with Human Pluripotent Stem Cell-Derived Cardiac Progenitors Reveals a Differentiation Stage-Specific Enhancement of Cardiomyocyte Maturation. Biotechnology journal, 14(8), e1800725.

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Tumor and Spleen Sample Preparation

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Tumors and spleens were removed with scissors or forceps and weighted. Tumors were chopped into fine pieces and transferred into RPMI media (5% FBS + 10 mM HEPES) containing collagenase (0.2 mg/mL) (Clostridium histolyticum, Sigma) and spleens were suspended in PBS. Tumors were shaken gently at 37 °C for 30 min prior to tissue disruption. Spleen and tumor tissues were disrupted using a cell strainer (70 µm nylon) (Corning) using a syringe plunge. Nylon mesh was rinsed several times with media. Samples were spin (5 min at 1500 rpm) and re-suspended in 1 mL RBC lysis buffer (Sigma) for 5 min and washed 2× with flow buffer (Thermo Fisher). Cells were counted (Nexelom Cellometer), prior to live/dead staining with Zombie UV (Biolegend), incubated for 15 min at room temperature and washed 2× with PBS. This was followed by Fc block (CD16/CD32, BD Biosciences), antibody staining and preparation of single-color compensation controls (Ultra Comp eBeads compensation beads, Thermo Fisher). Samples were covered in foil and kept at 4 °C overnight. Intracellular staining was performed using Permeabilization Buffer (eBioscience) in conjunction with Foxp3/Transcription Factor Staining Buffer set (eBioscience). Fluorescence minus one (FMO) controls were used where indicated to distinguish between positively and negatively stained cells for FoxP3, Ly6G, and Ly6C.

Gonzalez C., Williamson S., Gammon S.T., Glazer S., Rhee J.H, & Piwnica-Worms D. (2023). TLR5 agonists enhance anti-tumor immunity and overcome resistance to immune checkpoint therapy. Communications Biology, 6, 31.

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Isolation and Characterization of Retinal Cells

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Retinas were collected and digested in the same way as the steps previously mentioned. Immediately after the termination of digestion, the centrifuge tube containing the digested retina was cooled in ice and then centrifuged at 500×g at 4°C for 5 min. After the supernatant was discarded, the pellet was resuspended in 500 μL of precooled HBSS. Subsequently, the resuspended cell suspension was centrifuged at 800×g at 4°C for 5 min; and repeated step 1 time. After the supernatant was discarded again, the pellet was resuspended in 100 μL of flow buffer (eBioscience, USA). The classification of gating was based on Isotype Control which was equivalent to the negative control of the experiment. Thus, we chose Mouse IgG2A for gating. The flow antibody anti-rat CD11b/c (0.125 μg/tube, Mouse IgG2A, eBioscience) was added to the sample tube; the flow antibody Mouse IgG2A (0.125 μg/tube, eBioscience, USA) was added to the control tube. After mixing gently, the tubes were placed in the dark at 4°C for 30 min, and then centrifuged at 4°C and 300×g for 5 min. After the supernatant was discarded, the stained cell pellet was resuspended in 100 μL of flow buffer and centrifuged at 4°C and 300×g for 5 min, and this step was repeated once more. The stained cell pellet was resuspended in flow buffer, and then placed on ice before analyzed with flow cytometry.

Zhong H., Yu H., Sun J., Chen J., Huang S., Huang P., Liu X, & Zhong Y. (2021). Isolation of microglia from retinas of chronic ocular hypertensive rats. Open Life Sciences, 16(1), 992-1001.

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