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Precision count beads

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Precision Count Beads are monodisperse polymer microspheres used for accurate cell enumeration. They provide a reference standard for determining the absolute count of a target population in a sample.

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Lab products found in correlation

Lsrfortessa, by BD (6 mentions) Cd45 bv510, by BioLegend (4 mentions) Dnase 1, by Merck Group (4 mentions) Lsr fortessa cytometer, by BD (4 mentions) Lsr 2, by BD (3 mentions) Zombie nir fixable viability kit, by BioLegend (3 mentions) Pharm lyse buffer, by BD (3 mentions) Cd11c apc, by BD (3 mentions) Fc block, by BD (3 mentions) Recombinant human cxcl13, by R&D Systems (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Facsdiva software, by BD (3 mentions) True nuclear transcription factor buffer set, by BioLegend (3 mentions) Siglecf alexa647, by BD (2 mentions) Cd11c percp cy5, by BD (2 mentions) Ly 6c alexafluor700, by BD (2 mentions) Fix lyse solution, by Thermo Fisher Scientific (2 mentions) Cd45 pe fluor61, by Thermo Fisher Scientific (2 mentions) Cd11b pe cy7, by BD (2 mentions)

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Precision cell count beads Precision beads

114 protocols using precision count beads

1

Cytotoxicity Assay of Expanded Lymphocytes

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The 14-day DC/PC-3/IL-2-expanded lymphocytes were reconstituted and cultured (37 °C, 5% CO2) for 18–24 h in LM medium with or without IL-2 (80 IU/mL). The cells were pelleted, washed, and resuspended in fresh LM medium with or without IL-2 (80 IU/mL), and stimulated by coculturing with fluorescent TagFP635-PC-3 cells [39 (link)] at a ratio of 20:1 (lymphocytes:TagFP635-PC-3 cells). After 5-h coculturing, the cells were supplemented with Precision Count Beads (BioLegend), and the relative proportion of TagFP635-PC-3 cells to the Precision Count Beads determined by flow cytometry as described above. The cytotoxic activity was calculated as the difference between the relative proportion of TagFP635-PC-3 cells cocultured and not cocultured with DC/PC-3/IL-2-expanded lymphocytes.
Taborska P., Stakheev D., Svobodova H., Strizova Z., Bartunkova J, & Smrz D. (2020). Acute Conditioning of Antigen-Expanded CD8+ T Cells via the GSK3β-mTORC Axis Differentially Dictates Their Immediate and Distal Responses after Antigen Rechallenge. Cancers, 12(12), 3766.
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2

Multiparametric Flow Cytometry Analysis

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Cells from recipients’ PB and spleen were stained with antibodies described in Tables S3 and S4. For intracellular analysis of cytokine expression, spleen cell suspensions were stimulated with 1X Cell stimulation Cocktail plus protein transport inhibitors (Thermo Fisher Scientific) for 12-14h. Cells were fixed and intracellularly stained with antibodies described in Table S5 following BD Pharmingen™ Transcription Factor Buffer Set (BD Biosciences) instructions. Precision Count Beads (Biolegend) were added to peripheral blood and spleen prior to acquisition and the absolute numbers were calculated following the manufacturer’s instructions.
Hervás-Salcedo R., Fernández-García M., Hernando-Rodríguez M., Suárez-Cabrera C., Bueren J.A, & Yáñez R.M. (2023). Improved efficacy of mesenchymal stromal cells stably expressing CXCR4 and IL-10 in a xenogeneic graft versus host disease mouse model. Frontiers in Immunology, 14, 1062086.
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3

Characterization of Murine Lung Immune Cells

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Cells in BALF were stained with fixable viability dye and anti-murine CD45-PE Fluor61 (eBioscience, San Diego, CA), SiglecF-Alexa647, CD11b-PE Cy7 (BD Bioscience, Franklin Lakes, NJ), CD11c-PerCP Cy5.5, Ly-6C AlexaFluor700 (BD Pharmingen, San Jose, CA), and Ly-6G-FITC (Biolegend, San Diego, CA). Cells were fixed and red blood cells were removed using fix/lyse solution according to manufacturer’s instruction (eBioscience, San Diego, CA). Quantification of total cell content was performed by addition of precision count beads (Biolegend, San Diego, CA) before measurement. Expression levels of activation markers were quantified via the median fluorescent intensity (MFI). Data were collected on a Canto II flow cytometer (BD Bioscience, Franklin Lakes, NJ) and analyzed using FlowJo software v10.7.1 (Treestar, Palo Alto, CA, USA). The full gating strategy can be found in Fig. S1.
Meijer M.T., de Vos A.F., Peters Sengers H., Scicluna B.P., Roelofs J.J., Abou Fayçal C., Uhel F., Orend G, & van der Poll T. (2021). Tenascin C Has a Modest Protective Effect on Acute Lung Pathology during Methicillin-Resistant Staphylococcus aureus-Induced Pneumonia in Mice. Microbiology Spectrum, 9(1), 10.1128/spectrum.00207-21.
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4

Comprehensive Immune Cell Profiling in BAL

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Absolute cell counts in BAL were determined by flow cytometry with Precision Count Beads (BioLegend) by staining 100 μL of BAL fluid with pre-titrated amounts of CD45 FITC (clone HI30), CD14 PerCP-Cy5.5 (clone M5E2), CD3 PE-Cy7 (clone UCHT1), CD8 BV421 (clone RPA-T8), CD4 APC-Cy7 (clone OKT4) and CD19 APC (clone HIB19). 1 mL of FACS Lysing Solution (BD Biosciences) was added to each sample. 100 μL of Precision Count Beads were added immediately prior to flow cytometry to allow quantification of absolute volume analyzed on the cytometer.
In a separate experiment, fresh BAL cells were analyzed using a panel of antibodies directed against the following antigens: CD3 FITC (clone UCHT1), CD4 APC-Cy7 (clone OKT4), CD8 BV421 (clone RPA-T8), CD14 APC (clone M5E2), CD16 BV570 (clone 3G8), CD19 BV750 (clone HIB19), CD20 Pacific Blue (clone 2H7), CD38 PE-Cy7 (clone HIT2), CD45 Alexa Fluor 532 (clone HI30), CD56 PE/Dazzle 594 (clone HCD56), and HLA-DR BV605 (clone L243). 500,000–1,000,000 fresh BAL cells were stained in BD Brilliant Buffer (BD Biosciences) with Zombie NIR Fixable Viability Marker (BioLegend). Samples were run on a Cytek Aurora spectral flow cytometer using SpectroFlo software (version 2, Cytek) before final analysis in FlowJo software (version 10, BD Biosciences).
Reynolds D., Guillamet C.V., Day A., Borcherding N., Guillamet R.V., Choreño-Parra J.A., House S.L., O’Halloran J.A., Zúñiga J., Ellebedy A.H., Byers D.E, & Mudd P.A. (2021). Comprehensive immunologic evaluation of bronchoalveolar lavage samples from human patients with moderate and severe seasonal influenza and severe COVID-19. Journal of immunology (Baltimore, Md. : 1950), 207(5), 1229-1238.
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5

Quantifying Expanded B and T Cells

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All flow cytometry data were analyzed by using FlowJo (BD Biosciences). All relevant gating strategies are illustrated in supplemental Figures 1, 2, 3, and 4. Precision count beads (catalog #424902, BioLegend) were used to quantify absolute cell numbers. The percentage of viable B cells and the x-fold expansion of T cells represent absolute numbers of B or T cells normalized to the mean absolute number of B or T cells of untreated controls.
Roider T., Brinkmann B.J., Kim V., Knoll M., Kolb C., Roessner P.M., Bordas M., Dreger P., Müller-Tidow C., Huber W., Seiffert M, & Dietrich S. (2021). An autologous culture model of nodal B-cell lymphoma identifies ex vivo determinants of response to bispecific antibodies. Blood Advances, 5(23), 5060-5071.
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6

Cytotoxicity Assay of T Cell-Mediated Cancer Cell Killing

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Co-culture of cancer cells and T cells was performed as previously described (66 (link)). Briefly, B16F10 cells were maintained in complete DMEM media (10% FBS and 50U/ml of Penicillin-Streptomycin). CD8 T cells isolated from spleen and lymph nodes from Pmel-1 or OT-I mice were stimulated with anti-CD3/CD28 beads (ThermoFisher, 11452D) and then cultured in complete RPMI 1640 media (10%FBS, 20mM HEPES, 1mM sodium pyruvate, 0.05mM 2-mercaptoethanol, 2mM L-glutamine, and 50U/ml streptomycin and penicillin, 20ng/ml recombinant mouse IL-2).
To test the sensitivity of cancer cells to T cell-driven cytotoxicity (OT-I or Pmel-1 model), we plated B16F10 cells (sgRosa26, sgTraf3, pEF1a-Empty, or pEF1a-Traf3) at equal density in all wells, and added T cells at ratios to cancer cells. With the OT-I model, we first incubated the B16F10 cells with 1nM SIINFEKL peptide for 2 hours prior to co-culture with T cells. With the Pmel-1 model, B16F10 cells were either pre-treated with 1ng/ml IFNγ overnight or untreated prior to the co-culture. There are 2–4 cell-culture replicates for each condition. After a one-day or three-day co-culture with T cells, we counted the remaining cancer cells by FACS using the precision count beads (BioLegend, 424902). T cells present in these cultures were gated out based on antibodies specific for CD45 (Biolegend, clone 30-F11) or CD8 (BioLegend, clone 53-6.7).
Gu S.S., Zhang W., Wang X., Jiang P., Traugh N., Li Z., Meyer C., Stewig B., Xie Y., Bu X., Manos M.P., Font-Tello A., Gjini E., Lako A., Lim K., Conway J., Tewari A.K., Zeng Z., Sahu A.D., Tokheim C., Weirather J.L., Fu J., Zhang Y., Kroger B., Liang J.H., Cejas P., Freeman G.J., Rodig S., Long H.W., Gewurz B.E., Hodi F.S., Brown M, & Liu X.S. (2021). Therapeutically increasing MHC-I expression potentiates immune checkpoint blockade. Cancer discovery, 11(6), 1524-1541.
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7

Analyzing SARS-CoV-2-specific T-cell responses

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Splenocytes from vaccinated mice were cultured at 37°C with 5% CO2 for 24 hours in the presence of 5 SARS-CoV-2 specific peptide pools [5 ug/mL final concentration] in 96-wells U bottom plates at 1×106 PBMC per well. A stimulation with an equal percentage amount of DMSO was performed as a negative control while Concanavalin A (0.5 ug/mL final concentration) (eBioscience, Cat: 00–4978-93) was included as a positive control. Supernatants were harvested at 24 hours post-stimulation for various assays. Cells were washed and incubated with fluorochrome-conjugated antibodies for at least 15–20 min at 4 °C or on ice, protected from light. Precision count beads from Biolegend (Cat: 424902) were used to calculate absolute number of cells. The following fluorochrome-conjugated anti-mouse antibodies were used: CD3 (Clone: 17A2, Cat: 100216), CD4 (Clone: GK1.5; Cat: 100414), CD25 (Clone: PC61; Cat: 102010), OX40 (Clone: OX-86; Cat: 119411), PD-L1 (Clone: 10F.9G2; Cat: 124321), PD-1 (Clone: RMP1–30; Cat: 109110) were all from BioLegend. CXCR5 (Clone: 2G8; Cat: 560615) and 41BB (Clone: 1AH2; Cat: 558976) were from BD. LIVE/DEAD Fixable Dead Cell Stain (Life Technologies) (Cat: L34957) was used to gate on live cells. Samples were acquired on a BD LSR II and data were analyzed using FlowJo software (Treestar).
Cusimano G.M., Gary E.N., Bell M.R., Warner B.M., Connors J., Tursi N.J., Ali A.R., Zhang S., Canziani G., Taramangalam B., Gordon E.A., Chaiken I.M., Wootton S.K., Smith T., Ramos S., Kobasa D., Weiner D.B., Kutzler M.A, & Haddad E.K. (2022). Improved Durability to SARS-CoV-2 Vaccine Immunity Following Co-Immunization with Molecular Adjuvant Adenosine Deaminase-1. Journal of immunology (Baltimore, Md. : 1950), 209(1), 118-127.
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8

Immunophenotyping of Hematopoietic Stem and Progenitor Cells

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MNCs obtained as described above were stained for surface markers for 30 minutes at room temperature in the dark. An extended table describing the monoclonal antibodies used for immunophenotype analysis of hematopoietic stem and progenitor cell (HSPC) and lymphocyte subsets is provided in the Supplementary Data. Propidium iodide or Zombie Aqua viability dye (catalog no. 423101; BioLegend, San Diego, CA) were used to exclude dead cells. Data were collected on a BD FACSAria II (BD Biosciences, San Jose, CA) and analyzed with FlowJo version 9.9.4 (FlowJo Software, Ashland, OR). Precision count beads (catalog no. 424902; BioLegend) were used to obtain absolute cell counts for CD34+ cells, HSCs, and lymphocyte subpopulations according to the manufacturer's instructions. In brief, 100 µL of Precision count beads was added to 200 µL of sample, and absolute cell counts/µL were calculated using the following equation: (cell count × bead volume)/(bead count × cell volume) × bead concentration/µL.
Bankova A.K., Caveney J., Yao B., Ramos T.L., Bögeholz J., Heydari K., Diaz N., Jackson M.L., Lowsky R., Brown J.(., Johnston L., Rezvani A.R., Frank M.J., Muffly L., Weng W.K., Sidana S., Negrin R.S., Miklos D.B., Shiraz P., Meyer E.H., Shizuru J.A, & Arai S. (2022). Real-World Experience of Cryopreserved Allogeneic Hematopoietic Grafts during the COVID-19 Pandemic: A Single-Center Report. Transplantation and Cellular Therapy, 28(4), 215.e1-215.e10.
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9

Erythroid Progenitor Cell Culture

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FACS-isolated BFU-E enriched cells were cultured in progenitor culture media (PCM) consisting of serum free expansion media (SFEM II, Stem Cell Technologies) supplemented with 100 ng/mL murine SCF (Peprotech), 40 ng/mL murine insulin-like growth factor 1 (Peprotech), 2 U/mL human erythropoietin (Amgen), 1% penicillin/streptomycin (Gibco), and for indicated cultures 100 nM Dexamethasone (Sigma). Cells were seeded at known quantities and cultured at 37 degrees Celsius. Live cell numbers were counted at indicated days by both hemocytometer using Trypan Blue (Gibco) for dead cell exclusion, and by FACS on a FACS Fortessa cytometer (BD) normalizing to Precision Count Beads (Biolegend) and using Propidium Iodide (Sigma) for dead cell exclusion.
Li H., Natarajan A., Ezike J., Barrasa M.I., Le Y., Feder Z.A., Yang H., Ma C., Markoulaki S, & Lodish H.F. (2019). Rate of progression through a continuum of transit-amplifying progenitor cell states regulates blood cell production. Developmental cell, 49(1), 118-129.e7.
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10

Expansive Activation of γδT Cells

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In order to induce activation of γδT cells, PBMCs were incubated with 10 µg/ml PAM (Sigma-Aldrich, St Louis, USA) in 10% FBS RPMI-1640 plus 25 ng/ml recombinant human IL-2 (Peprotech, Rehovot, Israel) and/or 10 ng/ml recombinant human IL-15 (Peprotech, Rehovot, Israel) at 37 °C humidified cell incubator with 5% CO2. Half of the culture medium was replaced by a fresh medium and recombinant cytokines were added every 3 days. The purity of γδT cells was examined on days 0, 4, 7, 9, and 14 of culture by flow cytometry analysis. Then cell counting was determined by Precision Count Beads™ (BioLegend, San Diego, CA, USA). Only those cells under a 14-day culture presented with a ratio >90% γδ-TCR and CD3-positive cells were considered as a successful expansion and pure enough for further experiments.
For some cultures, γδT cells were pre-treated with AT-7519 (Selleck Chemicals, Houston, TX, USA) for 24 h before further culture in a regular culture medium devoid of cytokines. Cells were then harvested for corresponding assays.
Wang H., Wang X., Wang W., Chai W., Song W., Zhang H., Mou W., Pei M., Su Y., Ma X, & Gui J. (2022). Interleukin-15 enhanced the survival of human γδT cells by regulating the expression of Mcl-1 in neuroblastoma. Cell Death Discovery, 8, 139.
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