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Optilyse c

Manufactured by Beckman Coulter
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Sourced in United States, France

The Optilyse C is a laboratory instrument designed for cell analysis. It is used to prepare and analyze cell samples for subsequent flow cytometry.

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Lab products found in correlation

Phosphate buffered saline (pbs), by Cambrex (2 mentions) Facscanto flow cytometer, by BD (2 mentions) Gallios flow cytometer, by Beckman Coulter (2 mentions) 4 6 diamidino 2 phenylindole dihydrochloride, by Dojindo Laboratories (2 mentions) Facs lyse, by BD (2 mentions) Facscanto 2 cytometer, by BD (2 mentions) Vacutainer, by BD (2 mentions) Cd62l, by BD (2 mentions) Facsymphony, by BD (2 mentions) Bz x700 all in one fluorescence microscope, by Keyence (2 mentions) Lsm 700, by Zeiss (2 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) Cd3 pe cy7 clone ucht1, by Beckman Coulter (1 mentions) Cd45 pacific blue clone hi30, by BioLegend (1 mentions) Cytomics fc500, by Beckman Coulter (1 mentions) Flow cytometry, by Beckman Coulter (1 mentions) Rabbit anti human fibrinogen igg, by Agilent Technologies (1 mentions) Alexa fluor 594, by Thermo Fisher Scientific (1 mentions) Anti cd14 apc, by BD (1 mentions) Anti cd163 fitc, by BD (1 mentions)

32 protocols using optilyse c

1

Flow Cytometry Analysis of Immune Cells

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To get a single cell suspension, lymph nodes and spleens were put through a 40 µm filter. White blood cell counts of blood were measured on a Cell-Dyn 3200 automatic cell counter (Abbott Diagonstics).
Fifty µL of blood, spleen or lymph node cell suspension was added to 100 µL of antibody mixture containing different cell surface markers (supplemental table I) and incubated for 30 min in the dark at RT. After red blood cell lysis using Optilyse C (Beckman Coulter)10 minutes at RT, the samples were measured on the flow cytometer (Gallios, Beckman Coulter, Marseille, France). Kaluza Analysis Software 1.2 was used to analyze the data. Percentage of total cell number of neutrophils, monocytes and T cells as assessed by flow cytometry are presented.
de Haan J.J., Bosch L., Borgman A., Bastemeijer M., Brans M.A., van de Weg S.M., de Kleijn D.P., Sluijter J.P., el Azzouzi H, & de Jager S.C. (2017). Complement 5a Receptor deficiency does not influence adverse cardiac remodeling after pressure-overload in mice. Scientific Reports, 7, 17045.
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2

Peripheral Blood Immune Profiling

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Immuno-phenotyping of peripheral blood drawn into EDTA-containing blood collection tubes was performed using a modification of the Centers for Disease Control and Prevention guidelines in a Clinical Laboratory Improvement Act-certified laboratory, as previously described [16 (link)]. Cells were stained with combinations of monoclonal antibodies and then lysed with Optilyse-C (Beckman Coulter, Hialeah, FL), washed twice, and re-suspended in 500 μl of phosphate-buffered saline (Cambrex, Walkersville, MD). Samples were analyzed immediately on a Becton Dickinson FacsCanto flow cytometer (BDBiosciences, San Jose, CA). 4-color antibody panels used for cellular identification and enumeration have been previously described [16 (link)].
Lymphocytes were identified by side scatter and CD45 staining, with confirmation that less than 5% of cells within the lymphocyte gate expressed CD14 [18 (link)]. Absolute concentrations of specific lymphocyte populations (CD3+, CD4+, CD8+, CD4+/8+, CD19, and CD16+/56+ lymphocytes) were calculated using the measured percentage of each cell type within the lymphocyte gate and the absolute lymphocyte concentration [16 (link)].
Orr C., Xu W., Masur H., Kottilil S, & Meissner E.G. (2020). Peripheral blood correlates of virologic relapse after Sofosbuvir and Ribavirin treatment of Genotype-1 HCV infection. BMC Infectious Diseases, 20, 929.
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3

Multiparameter Flow Cytometry of Immune Cells

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Whole blood was stained with antibodies and fixed with Optilyse C (Beckman Coulter, Krefeld, Germany) according to the manufacturer's instructions. Data were acquired on a Gallios flow cytometer (Beckman Coulter) and analyzed using FlowJo software (version 10; Treestar, Ashland, OR, USA). Antibodies were: CD4 PerCP (clone SK3), CXCR5 FITC (clone RF8B2) (all from BD Biosciences, Heidelberg, Germany), CD3 PE-Cy7 (clone UCHT1, Beckman Coulter), CD45 Pacific Blue (clone HI30), CD45RA APC-Cy7 (clone HI100), CCR6 BV605 (clone G034E3) (all from BioLegend, San Diego, CA, USA), CXCR3 PE (clone WM59, R&D Systems, Minneapolis, MN, USA).
Klemann C., Camacho-Ordonez N., Yang L., Eskandarian Z., Rojas-Restrepo J.L., Frede N., Bulashevska A., Heeg M., Al-Ddafari M.S., Premm J., Seidl M., Ammann S., Sherkat R., Radhakrishnan N., Warnatz K., Unger S., Kobbe R., Hüfner A., Leahy T.R., Ip W., Burns S.O., Fliegauf M, & Grimbacher B. (2019). Clinical and Immunological Phenotype of Patients With Primary Immunodeficiency Due to Damaging Mutations in NFKB2. Frontiers in Immunology, 10, 297.
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4

Multiparametric Immunophenotyping of Peripheral Blood

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Peripheral blood was collected via either tail vein puncture or cardiac puncture. Fetal bovine serum (FBS, 2%; Thermo Fisher Scientific, Waltham, MA, USA) in PBS was combined with heparin-added peripheral blood with the antibody reagent described below. The mixture was allowed to react in a cool and dark place for 30 min. After this reaction, OptiLyse® C (Beckman Coulter, Brea, CA, USA) was added, and then hemolysis and fixation were performed in the dark at room temperature (20°C–25 °C) for 15 min. After hemolysis and fixation, the sample was washed with PBS, and the cell suspension was analyzed using Cytomics FC 500 (Beckman Coulter). The following antibodies were used: anti-human CD3-phycoerythrin-Texas Red-x (ECD, Clone: UCHT1, Beckman Coulter), anti-human CD4-r-phycoerythrin (PE, clone: RPA-T4, Becton Dickinson, San Jose, CA, USA), anti-human CD8-fluorescein isothiocyanate (FITC, clone: RPA-T8, Becton Dickinson), anti-human CD19-phycoerythrin-Cy7 (PC7, Clone: J3-119, Beckman Coulter), anti-human CD34-PE (clone: 581, BD Pharmigen™, San Jose, CA, USA), and anti-human CD45-phycoerythrin-Cy5 (PC5, Clone: J33, Beckman Coulter).
Tsuzuki H., Nagatsuka Y., Iwata M., Kitamura N., Nagasawa Y., Matsumoto T., Ito R., Takahashi T., Ito M., Nakamura H, & Takei M. (2021). Antinuclear antibodies produced in HLA-DR transgenic humanized mice developed chronic graft-versus-host disease. Heliyon, 7(11), e08380.
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5

Flow Cytometry Analysis of Immune Cells

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Blood and bone marrow were collected 12 weeks after radiation. Cells were incubated with anti-rat CD3-FITC/CD45RA-PC7 and anti-rat CD3-FITC/CD4-PC7/CD8-APC (Beckman Coulter, High Wycombe, UK) at room temperature for 20 min, then with OptiLyse C (Beckman Coulter, UK) at room temperature for 10 min. The cells were then washed twice with phosphate-buffered saline (PBS) and resuspended in 500 μl PBS. All samples were assessed by flow cytometry (Beckman Coulter, Brea, CA, USA). The percentage of cells was based on the evaluation of 100,000 events for each culture condition [19 (link)].
Xu X., Li R., Zhou Y., Zou Q., Ding Q., Wang J., Jin W., Hua G, & Gao J. (2017). Dysregulated systemic lymphocytes affect the balance of osteogenic/adipogenic differentiation of bone mesenchymal stem cells after local irradiation. Stem Cell Research & Therapy, 8, 71.
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6

Thrombus Composition Analysis by IF

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Immediately after the T-TAS analysis, immunofluorescence analysis was performed to determine the composition of thrombi formed in the flow chamber [22 (link)]. Platelets in unfixed thrombi were labeled with FITC-conjugated mouse anti-human CD41 IgG (Beckman Coulter, Miami, FL, USA) for 15 min in the dark. After fixation with OptiLyse C (Beckman Coulter), fibrin (ogen) was detected by using rabbit anti-human fibrinogen IgG (Dako, Tokyo, Japan) labeled with Alexa Fluor 594 (Invitrogen, Carlsbad, CA, USA) for 30 min in the dark. The nuclei of leukocytes were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (Dojindo, Kumamoto, Japan). The entire image of thrombi formed in the flow chamber was analyzed with a BZ-X700 All-in-One Fluorescence Microscope (Keyence Corp., Osaka, Japan). Although fibrin generation could continue until fixation with OptiLyse C, this had little impact on the results.
Atari B., Ito T., Nagasato T., Ohnishi T., Hosokawa K., Yasuda T., Maruyama I, & Kakihana Y. (2020). A modified microchip-based flow chamber system for evaluating thrombogenicity in patients with thrombocytopenia. Thrombosis Journal, 18, 31.
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7

Leukocyte Surface Marker Analysis

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The authors collected blood samples from the antecubital fossa vein. For the surface staining of leukocytes, 50 µL of anticoagulated blood was added to tubes containing 5 µL of fluorochrome-labeled monoclonal antibodies, including anti-CD11b allophycocyanin (APC), clone Bear1; anti-CD14-APC, clone M5E2; anti-CD15 fluorescein isothiocyanate (FITC), clone 80H5; anti-CD16 R-phycoerythrin (RPE), clone 3G8; anti-CD62L-RPE, clone DREG56; anti-CD64-RPE, clone 22; anti-CD163-FITC, clone GHI/61; and anti-CD274-APC, clone PD-L1. The anti-CD163-FITC and anti-CD14-APC were manufactured by BD Biosciences (Franklin Lakes, NJ, USA), and all the other antibodies were manufactured by Beckman Coulter (Miami, FL, USA). The blood samples were incubated with the antibodies for 15 min at room temperature in the dark. Then, a lysing solution (OptiLyse C, Beckman Coulter, Miami, FL, USA) was added, and the mixture was incubated for another 10 min. The flow cytometric evaluation was conducted with a Navios 10 flow cytometer (Beckman Coulter, Miami, FL, USA). The gating strategy for the granulocytes and monocytes is presented in Figure 1.
Pavelek Z., Angelucci F., Souček O., Krejsek J., Sobíšek L., Klímová B., Šarláková J., Halúsková S., Kuča K, & Vališ M. (2020). Innate Immune System and Multiple Sclerosis. Granulocyte Numbers Are Reduced in Patients Affected by Relapsing-Remitting Multiple Sclerosis during the Remission Phase. Journal of Clinical Medicine, 9(5), 1468.
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8

Identification of OVA-specific CD8+ T cells

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A portion of the collected peripheral blood was treated with EDTA/3K. A 10 μL volume of T-Select H-2Kb OVA Tetramer-SINFEKL-phycoerythrin (PE) (TS-5001-1C; Medical & Biological Laboratories, Nagoya, Japan) was added to 50–100 μL EDTA-treated blood, and the mixture was allowed to stand at 25°C for 20 min before adding 1 μL of mouse anti-CD3 PE-cyanine 5-conjugated antibody (Thermo Fisher Scientific) and 1 μL of mouse anti-CD8 FITC-conjugated monoclonal antibody (KT15) (Medical & Biological Laboratories). After 20 min, 500 μL Optilyse C (Beckman Coulter, Brea, CA, USA) was added to the blood to induce hemolysis, and the mixture was allowed to stand for 10 min before adding 500 μL PBS. The cell suspension was purified on a cell sorter (SH800; Sony, Tokyo, Japan). To identify OVA-specific CD8+ T cells, lymphocytes were gated on the basis of a forward scatter (FSC)-vs-side scatter (SSC) density plot, and then developed to SSC-vs-CD3 density plot. The CD3+ population was further developed into an OVA tetramer-vs-CD8 density plot.
To obtain serum, peripheral blood was centrifuged at 1,800× g for 15 min at 4°C. The levels of OVA-specific IgG1 and IgG2a were assayed using mouse anti-OVA IgG1 and IgG2a assay kits (both from Chondrex, Redmond, WA, USA).
Hanagata N., Li X., Chen M.H., Li J, & Hattori S. (2017). Double-stranded phosphodiester cytosine-guanine oligodeoxynucleotide complexed with calcium phosphate as a potent vaccine adjuvant for activating cellular and Th1-type humoral immunities. International Journal of Nanomedicine, 13, 43-62.
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9

Multicolor Flow Cytometry Analysis of Blood

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An amount of 100 µL of fresh Na heparin anti-coagulated peripheral blood samples (<4 h after venesection) were stained with monoclonal antibodies (mAb) according to the manufacturers’ directions, incubated for 15 min at RT, lysed with Optilyse C (Beckman Coulter, Brea, CA, USA) for 10 min at RT, washed with 2 mL PBS and resuspended in 250 µL 0.5% paraformaldehyde in PBS, as previously described [12 (link),37 (link)]. Intracellular staining for cytotoxic granules and granzymes was performed using FACSLyse (BD Biosciences, San Jose, CA, USA) and FACSPerm 2 (BD Biosciences) as previously described [12 (link)]. The mAb’s used in this study are listed in Supplementary Table S3. Fixed samples were kept at 4 °C before 14-colour analysis, within 24 h, on an LSR II (BD Biosciences) as previously described [12 (link),37 (link),38 (link)].
Frith K., Munier C.M., Hastings L., Mowat D., Wilson M., Seddiki N., Macintosh R., Kelleher A.D., Gray P, & Zaunders J.J. (2021). The Role of ZEB2 in Human CD8 T Lymphocytes: Clinical and Cellular Immune Profiling in Mowat–Wilson Syndrome. International Journal of Molecular Sciences, 22(10), 5324.
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10

Peripheral Blood Immune Cell Analysis

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Patient blood samples were collected by venipuncture. All peripheral blood samples (5 ml per subject) were collected in vacutainer tubes (BD Biosciences, San Jose, CA, USA) containing ethylenediaminetetraacetic acid (EDTA). For surface staining, 100 µL of heparinized peripheral blood was added to tubes containing 10 µL of mouse anti-human monoclonal antibodies (mAbs), including Peridinin Chlorophyll Protein Complex (PerCP)-conjugated anti-CD45, FITC-conjugated anti-CD3, APC-conjugated anti-CD4, PE-conjugated anti-CD8, and PE-conjugated anti-CD16+CD56, which were provided by ACEA Biosciences (San Diego, CA, USA). Isotype-matched mouse anti-human IgG antibodies served as negative controls for all fluorescein-conjugated IgG mAbs. Thereafter, the blood was mixed with the cocktail monoclonal antibody solution and incubated for 15 min at room temperature. Then, a lysing solution (OptiLyse C, Beckman Coulter) was added, and the mixture was incubated for another 15 min. To detect the percentage and absolute cell numbers of different subsets in peripheral blood, cells were collected and analyzed on a NovoCyte Flow Cytometer (ACEA Biosciences, San Diego, CA, USA) using NovoExpress Software (ACEA Biosciences, San Diego, CA, USA).
Jiang H., Yu K., Cui Y., Ren X., Li M., Yang C., Zhao X., Zhu Q, & Lin S. (2021). Combination of Immunotherapy and Radiotherapy for Recurrent Malignant Gliomas: Results From a Prospective Study. Frontiers in Immunology, 12, 632547.
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