Single stained polystyrene beads

Manufactured by BD

Single-stained polystyrene beads are a type of lab equipment used for various applications. They are made of polystyrene and have a single fluorescent dye incorporated into their structure. These beads provide a consistent and uniform platform for various experiments and analyses.

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Lab products found in correlation

Lsrfortessa flow cytometer, by BD (6 mentions) Cytofix cytoperm, by BD (4 mentions) Perm wash, by BD (3 mentions) Trypsin edta, by Thermo Fisher Scientific (3 mentions) Transcription factor perm wash buffer, by BD (2 mentions) Transcription factor fixation permeabilization buffer, by BD (2 mentions) Live dead fixable near ir dead cell stain, by Thermo Fisher Scientific (1 mentions) Facsverse, by BD (1 mentions) Gentamicin, by Thermo Fisher Scientific (1 mentions) R version 3, by R Project for Statistical Computing (1 mentions) Normocin, by InvivoGen (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) Anti cd3 ecd, by Beckman Coulter (1 mentions) Live dead aqua, by Thermo Fisher Scientific (1 mentions) Anti ccr7 brilliant violet 421, by BioLegend (1 mentions) Anti ki67 fitc, by BD (1 mentions) Anti cd127 pe cy7, by BD (1 mentions) Anti cd25 pe cy7, by BD (1 mentions) Anti cd4 qdot655, by Thermo Fisher Scientific (1 mentions)

8 protocols using single stained polystyrene beads

Flow Cytometry Staining Protocol

Cited 2 times

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The antibodies used are listed in Supplemental Table 1. Cell surface and intracellular staining was performed as has been described previously [4 (link), 15 (link), 16 (link)]. Samples were acquired on an LSRFortessa flow cytometer (BD Biosciences) equipped with 405, 488, 561, and 639 nm lasers. Single-stained polystyrene beads (BD Biosciences) were used for compensation, which was performed using the compensation platform in FlowJo software v. 9.8 (Tree Star).

Dias J., Sobkowiak M.J., Sandberg J.K, & Leeansyah E. (2016). Human MAIT-cell responses to Escherichia coli: activation, cytokine production, proliferation, and cytotoxicity. Journal of Leukocyte Biology, 100(1), 233-240.

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Multiparametric Flow Cytometry Protocol

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Surface and intracellular stainings were performed as previously described (50 (link)). Cells were stained for surface markers for 20 min, followed by fixation and permeabilization with CytoFix/Cytoperm (BD Bioscience) for 30 min before intracellular staining for 30 min. The antibodies used are listed in SI Appendix, Table S1. All stainings were performed at 4 °C. Samples were acquired on an LSRFortessa flow cytometer (BD Biosciences) equipped with 355-, 405-, 488-, 561-, and 639-nm lasers. Compensation was performed using single-stained polystyrene beads (BD Biosciences), and the compensation platform in the FlowJo software v. 10.

Emgård J., Bergsten H., McCormick J.K., Barrantes I., Skrede S., Sandberg J.K, & Norrby-Teglund A. (2019). MAIT Cells Are Major Contributors to the Cytokine Response in Group A Streptococcal Toxic Shock Syndrome. Proceedings of the National Academy of Sciences of the United States of America, 116(51), 25923-25931.

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Flow Cytometric Analysis of MAIT Cell Effector Molecules

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Cell-surface staining was performed using directly-conjugated antibodies and cells were fixed in Cytofix/Cytoperm or in Transcription Factor Fixation/Permeabilization buffer (both from BD Biosciences) as appropriate. Intracellular staining was performed in Perm/Wash or Transcription Factor Perm/Wash buffer as appropriate (both from BD Biosciences). Samples were acquired on an LSRFortessa flow cytometer (BD Biosciences) equipped with 405, 488, 561, and 639 nm lasers. Single-stained polystyrene beads (BD Biosciences) were used for compensation purposes. Software-based compensation was performed using the compensation platform in FlowJo software version 9.6 (Tree Star, Ashland, OR, USA). Distribution of effector molecules expressed by MAIT cells was compared using SPICE software version 5.35.^{48 (link)} Antibodies used in the experiments are listed in Supplementary Table 2.

Gibbs A., Leeansyah E., Introini A., Paquin-Proulx D., Hasselrot K., Andersson E., Broliden K., Sandberg J.K, & Tjernlund A. (2016). MAIT cells reside in the female genital mucosa and are biased towards IL-17 and IL-22 production in response to bacterial stimulation. Mucosal immunology, 10(1), 35-45.

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Flow Cytometric Analysis of MAIT Cell Effector Molecules

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Quantifying DNA Damage Response

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After 24 h of co-culture at 37°C, 5% CO₂, the medium was aspirated from the wells and cell layer detached using trypsin-EDTA (Thermo Fisher Scientific). The cells were washed with FACS buffer (PBS + 2% FCS + 2 mM EDTA) and stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain (Thermo Fisher Scientific) for 20 minutes on ice. After washing away excess reagent with FACS buffer, the cells were stained with H2A.X Phosphorylation Assay Kit for Flow Cytometry (MilliporeSigma, St Louis, MO, USA), in accordance with the manufacturer’s instructions. Stained samples were acquired on a FACSVerse flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Single-stained polystyrene beads (BD Biosciences) were used for compensation. Flow cytometry data analysis was performed in FlowJo software v10.6.2 (BD).

Halimi A., Gabarrini G., Sobkowiak M.J., Ateeb Z., Davanian H., Gaiser R.A., Arnelo U., Valente R., Wong A.Y., Moro C.F., Del Chiaro M., Özenci V, & Chen M.S. (2021). Isolation of pancreatic microbiota from cystic precursors of pancreatic cancer with intracellular growth and DNA damaging properties. Gut Microbes, 13(1), 1983101.

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Phenotypic and Functional Analysis of PBMCs

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PBMCs were thawed and washed once in RPMI-1640 medium supplemented with 25 mM HEPES, 2 mM L-glutamine (all from Thermo Fisher Scientific), 10% FBS (Sigma-Aldrich), 50 μg/mL gentamicin (Life Technologies), and 100 μg/mL normocin (InvivoGen). PBMCs were then used for phenotypic stainings or functional assays. Surface and intracellular stainings for cytokines and transcription factors were performed as previously described.^{32 (link)} The antibodies used are listed in Supplementary Table S2 as well as in Supplementary CTAT Table. Samples were acquired on an LSRFortessa flow cytometer (BD Biosciences) equipped with 355, 405, 488, 561, and 639 nm lasers. Single-stained polystyrene beads (BD Biosciences) and the compensation platform in the FlowJo software v. 9.9 or v. 10 (TreeStar) were used for data compensation. Unless otherwise indicated, MAIT cells were defined as CD161^hiVα7.2⁺ cells being CD8⁺CD4⁻ or CD8⁻CD4⁻. High dimensional analysis of flow cytometry data was performed through Barnes-Hut stochastic neighbor embedding (SNE) analysis using R (version 3.2.1, The R Foundation for Statistical Computing) with an in-house built script as previously described.^{12 (link)}

Dias J., Hengst J., Parrot T., Leeansyah E., Lunemann S., Malone D.F., Hardtke S., Strauss O., Zimmer C.L., Berglin L., Schirdewahn T., Ciesek S., Marquardt N., von Hahn T., Manns M.P., Cornberg M., Ljunggren H.G., Wedemeyer H., Sandberg J.K, & Björkström N.K. (2019). Chronic hepatitis delta virus infection leads to functional impairment and severe loss of MAIT cells. Journal of hepatology, 71(2), 301-312.

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Detailed Phenotyping of Immune Cells

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Anti-CD14 and anti-CD19 APC-H7 (both at 1:100), anti-CD25 PECy7 (1:50), anti-CD62L V450 (1:50), anti-CD127 PECy7 (1:100), anti-CD161 FITC (1:10), anti-IFNγ V450 (1:200), anti-Ki67 FITC (1:10) were from BD Biosciences. Anti-CD3 ECD (1:100) and anti-CD8β PE (1:50) were from Beckman Coulter. Anti-Vα7.2 FITC (1:10) and PE (1:20), anti-CD45 Alexa Fluor 700 (1:400), anti-CD45RO PECy7 (1:50), anti-CCR9 Alexa Fluor 647 (1:20), anti-Ki67 Brilliant Violet 421 (1:20), anti-CCR7 Brilliant Violet 421 (1:20) were from BioLegend. Anti-CD161 PerCP-Cy5.5 (1:20), anti-IL-22 PerCP-eFluor 710 (1:50) were from eBioscience. Anti-IL-18R PE (1:20), and anti-PLZF APC (1:10) were from R&D systems. Anti-CD4 Qdot 655 (1:400), anti-CD8α Qdot 605 (1:800), anti-CD45 Qdot 705 (1:100), live/dead aqua (1:100) and near infrared fixable (1:400) cell stain were from Invitrogen. Cell surface staining was performed using directly conjugated antibodies and fixed in Cytofix/Cytoperm (BD Biosciences). Intracellular staining was performed using the appropriate mAb’s in Perm/Wash (BD Biosciences). Samples were acquired on an LSRII flow cytometer (BD Biosciences) equipped with 405, 488, 532 and 647 nm lasers. Single-stained polystyrene beads (BD Biosciences) were used for compensation purposes. Software-based compensation was performed using the compensation platform in FlowJo software version 9.5 (Tree Star).

Leeansyah E., Loh L., Nixon D.F, & Sandberg J.K. (2014). Acquisition of innate-like microbial reactivity in mucosal tissues during human fetal MAIT-cell development. Nature Communications, 5, 3143.

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Comprehensive Immunophenotyping Assay for Adaptive Immunity

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Cell surface and intracellular staining for cytokines, cytotoxic molecules, and active Casp3 were performed as previously described [2 (link)]. Staining with the MR1 5-OP-RU and MR1 6-FP tetramers was performed for 40 min at room temperature (RT) [7 (link)] before proceeding to the surface and intracellular staining with other mAbs (S3 Table.) Samples were acquired on an LSRFortessa flow cytometer (BD Biosciences) equipped with 355, 405, 488, 561, and 640 nm lasers. Single-stained polystyrene beads (BD Biosciences) and the compensation platform in FACSDiva version 8.0.1 (BD Biosciences) or FlowJo software versions 9.9 and 10.5 (TreeStar) were used for compensation.

Boulouis C., Sia W.R., Gulam M.Y., Teo J.Q., Png Y.T., Phan T.K., Mak J.Y., Fairlie D.P., Poon I.K., Koh T.H., Bergman P., Lim C.M., Wang L.F., Kwa A.L., Sandberg J.K, & Leeansyah E. (2020). Human MAIT cell cytolytic effector proteins synergize to overcome carbapenem resistance in Escherichia coli. PLoS Biology, 18(6), e3000644.

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