Perfectcount beads

Manufactured by Cytognos

Sourced in Spain

PerfectCount beads are a type of calibration beads used in flow cytometry. They are designed to provide accurate and consistent cell counting results. The beads have a known concentration and can be used to calibrate flow cytometers, ensuring reliable and reproducible cell enumeration.

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Lab products found in correlation

Facs lysing solution, by BD (1 mentions) Facscanto 2, by Tree Star (1 mentions) Zombie nir fixable viability kit, by BioLegend (1 mentions) Cd45.1 clone a20, by BioLegend (1 mentions) Easysep human cd14 positive selection kit, by STEMCELL (1 mentions) Phycoerythrin pe conjugated annexin 5, by Immunotools (1 mentions) Facsdiva software, by BD (1 mentions) Facscanto 2 flow cytometer, by BD (1 mentions) 7 aminoactinomycin d 7 aad, by BD (1 mentions)

4 protocols using perfectcount beads

Lymphocyte Enumeration Protocol

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Quantification of lymphocyte numbers procedure was previously described (23 (link)). Briefly, 25 µl of peripheral blood samples were incubated with CD45-PerCP (Beckton Dickinson (BD) Biosciences, San José, CA, USA) for 15 minutes at room temperature and protected from the light. Erythrocytes were removed using 450 µl lysis buffer (BD FACS™ Lysing Solution, BD Biosciences). 25 µl of PerfectCount beads (Cytognos SL, Salamanca, Spain) were added to each sample and acquired on the same flow cytometer. The number of total lymphocytes was expressed in cells/µl.
Percentage and absolute counts (number of cells per µl) were analysed for all subpopulations defined in Table 1. Absolute counts were calculated as follows: (%subset/100) x counts of main subpopulations.

Teniente-Serra A., Pizarro E., Quirant-Sánchez B., Fernández M.A., Vives-Pi M, & Martinez-Caceres E.M. (2021). Identifying Changes in Peripheral Lymphocyte Subpopulations in Adult Onset Type 1 Diabetes. Frontiers in Immunology, 12, 784110.

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Cytotoxicity Evaluation of CAR-T Cells

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Real-time cytotoxicity assay (xCELLigence) was carried out to analyze the cytotoxicity of the CD8⁺ CAR-T cells as previously described19 (link) and using an Effector:Tumor cell ratio of 1:1). All experiments were performed in duplicate.
CAR-T cell cytotoxicity was also measured by flow cytometry. 6×10⁵ CAR-T cells were cocultured with PM299L-EDA^high PM299L-EDA^low or PM299L-Thy1.1 cells for 24 hours at two different Effector:Tumor ratios (1:1 and 0.2:1). Then, cells were washed and incubated with a fluorochrome-conjugated antibody against CD8. Perfect-Count beads (Cytognos) were added for the flow cytometric quantification of absolute cell numbers.

Martín-Otal C., Lasarte-Cia A., Serrano D., Casares N., Conde E., Navarro F., Sánchez-Moreno I., Gorraiz M., Sarrión P., Calvo A., De Andrea C.E., Echeveste J., Vilas A., Rodriguez-Madoz J.R., San Miguel J., Prosper F., Hervas-Stubbs S., Lasarte J.J, & Lozano T. (2022). Targeting the extra domain A of fibronectin for cancer therapy with CAR-T cells. Journal for Immunotherapy of Cancer, 10(8), e004479.

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Quantifying T-cell Activation and Migration

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OT1 T-lymphocytes activated for 48 h in the presence or absence of ICAM-1 blocking mAbs were stained with antibodies against the chemokine receptors CCR7 (clone 4B12; Biolegend). Single cell suspensions from tumors and lymph nodes were stained with antibodies against CD45.1 (clone A20, Biolegend) and counted using perfect count beads (Cytognos, Salamanca, Spain) as internal standards. Dead cells were excluded using the Zombie NIR Fixable viability kit (Biolegend). Cells were collected with FACSCanto II and cell acquisition data was analyzed using FlowJo software (Treestar, Ashland, OR).

Yanguas A., Garasa S., Teijeira Á., Aubá C., Melero I, & Rouzaut A. (2018). ICAM-1-LFA-1 Dependent CD8+ T-Lymphocyte Aggregation in Tumor Tissue Prevents Recirculation to Draining Lymph Nodes. Frontiers in Immunology, 9, 2084.

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Isolation and Characterization of Monocytes

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Healthy donor buffy coat samples were processed first depleting CD3⁺ cells using the RoseetteSep® Human Monocyte Enrichment Kit (StemCell Technologies, Vancouver, Canada) prior to a density gradient separation using ficoll-hypaque (Rafer, Zaragoza, Spain). Afterwards, CD14⁺ cells were isolated using the EasySep® Human CD14 Positive Selection Kit (StemCell), according to the manufacturer’s instructions. Cell viability was determined using 7-amino-actinomycin D (7-AAD) (BD Biosciences, Franklin Lakes, NK, USA) and phycoerythrin (PE)-conjugated annexin V (Immunotools, Friesoythe, Germany) staining for 20 min at 4°C, protected from light, and cell counts were quantified simultaneously using PerfectCount beads (Cytognos, Salamanca, Spain). Samples were acquired on a FACSCanto II flow cytometer (BD Biosciences), and monocyte purity was determined using forward and side scatter gating strategies on FACSDiva software (BD Biosciences).

Navarro-Barriuso J., Mansilla M.J., Quirant-Sánchez B., Teniente-Serra A., Ramo-Tello C, & Martínez-Cáceres E.M. (2021). Vitamin D3-Induced Tolerogenic Dendritic Cells Modulate the Transcriptomic Profile of T CD4+ Cells Towards a Functional Hyporesponsiveness. Frontiers in Immunology, 11, 599623.

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