Iotest 3 lysing solution

Manufactured by Beckman Coulter

Sourced in United States

IOTest 3 Lysing Solution is a laboratory reagent designed for use in flow cytometry applications. It is used to lyse red blood cells, allowing for the analysis of white blood cells and other cells of interest. The solution contains proprietary components that facilitate the lysis process, enabling the efficient preparation of samples for flow cytometric analysis.

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IOTest 3 (4 citations) Lysing Solution (2 citations)

6 protocols using iotest 3 lysing solution

Whole Blood Immune Response Profiling

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In the FASCIA method, diluted whole blood is stimulated for seven days with three mitogens ConA, phytohemagglutinin (PHA), and pokeweed mitogen (PWM) as described in detail before [8 (link)]. Minimal blood volume requirement for the test is 0.5 ml. We used the following mitogens and final concentrations in RPMI media (supplemented with 2 mM L-glutamine, HEPES, 100 IU/ml penicillin, and 100 IU/ml streptomycin): PHA 10 μg/ml, ConA 10 μg/ml, and PWM 5 μg/ml (all mitogens from Sigma-Aldrich, USA). PHA and ConA are primarily T cell mitogens, and PWM is widely used to stimulate B cells. Our laboratory has tested that samples can be stimulated after approximately 27 h storage or transport at room temperature without compromising the results (data not shown).
After the incubation, red cells were lysed with IOTest 3 Lysing Solution (Beckman Coulter, USA) and stained with following directly conjugated antibodies: CD8 PerCP-Cy5.5 (clone SK1), CD19 APC (clone SJ25C1), and BD Simultest CD3-FITC/CD4-PE (clones SK7 and SK3 respectively), all antibodies from BD Biosciences, USA. Flow cytometry data was acquired with FACS Canto equipment (BD Biosciences), and the data analyzed with FACS Diva software (BD Biosciences).

Lusila P., Toivonen A., Jarva H., Vettenranta K., Lehtimäki S, & Kekäläinen E. (2022). FASCIA Method in the Assessment of Lymphocyte Mitogen Responses in the Laboratory Diagnostics of Primary Immunodeficiencies. Journal of Clinical Immunology, 43(3), 653-661.

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Quantifying Chimerism Levels in Mice

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Chimerism levels of recipient mice were assessed by flow cytometry on a Cytomics FC500 (Beckman Coulter, USA) as the percentage of GFP-positive donor cells in mononuclear cell fraction and granulocyte fraction (Fig. 1). Peripheral blood was collected in heparinized capillary tubes by retroorbital sinus puncture at 4 and 8 weeks of age, and in some chimeric mice at every 8 weeks until 48 weeks of age. The blood was washed, and red blood cells were lysed using IOTest 3 Lysing Solution (Beckman Coulter, France). 7-AAD (Beckman Coulter, France) was used to exclude dead cells.

Ihara N., Akihiro U., Onami N., Tsumura H., Inoue E., Hayashi S., Sago H, & Mizutani S. (2015). Partial rescue of mucopolysaccharidosis type VII mice with a lifelong engraftment of allogeneic stem cells in utero. Congenital Anomalies, 55(1), 55-64.

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Immunophenotyping of Cell Populations

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The total cell numbers and cell viabilities were analyzed for samples collected pre- and post-selection, after medium exchange for pre-conditioning, and in the final product before cryopreservation. Flow cytometric analysis was performed for quantification of CD45⁺ hematopoietic cells, CD14⁺ monocytes, CD34⁺ stem cell progenitors, CD3⁺ T cells, CD19⁺ B cells, and CD56⁺ NK cells (the conjugated monoclonal antibodies are listed in Table S1). A maximum of 2 × 10⁶ cells were stained with the indicated volume of antibodies (see Table S1). The cells were then stained with 7-AAD for 15 min at RT for discrimination of dead cells. Afterward, the cells were incubated with freshly prepared IOTest3 Lysing Solution (Beckman Coulter) for 15 min at RT according to the manufacturer’s instructions. Prior to acquisition analyses by flow cytometry, FlowCount Beads (Beckman Coulter) were added to the samples according to the manufacturer’s instructions. Analyses were performed with the Navios flow cytometer (Navios 3L 10C, software 1.3; Beckman Coulter). Cell debris were excluded via forward-scatter (FSC)/side-scatter (SSC) scatterplot. Viable leukocytes were defined as CD45⁺ and 7-AAD^neg. Viable CD45⁺ leukocytes were further analyzed for the frequencies of CD34⁺, CD3⁺, CD19⁺, CD14⁺, and CD56⁺ cells (see gating strategy in Figure S7A).

Bialek-Waldmann J.K., Domning S., Esser R., Glienke W., Mertens M., Aleksandrova K., Arseniev L., Kumar S., Schneider A., Koenig J., Theobald S.J., Tsay H.C., Cornelius A.D., Bonifacius A., Eiz-Vesper B., Figueiredo C., Schaudien D., Talbot S.R., Bleich A., Spineli L.M., von Kaisenberg C., Clark C., Blasczyk R., Heuser M., Ganser A., Köhl U., Farzaneh F, & Stripecke R. (2021). Induced dendritic cells co-expressing GM-CSF/IFN-α/tWT1 priming T and B cells and automated manufacturing to boost GvL. Molecular Therapy. Methods & Clinical Development, 21, 621-641.

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Immunophenotyping of Chronic Lymphocytic Leukemia

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PBMCs were separated by density centrifugation of fresh CLL blood samples. PBMCs were incubated with directly conjugated mAbs for 20 minutes at room temperature. Flow cytometric analysis was performed after erythrolysis with IOTest® 3 Lysing Solution (Beckman Coulter, IM3514) and washing steps. Monoclonal antibodies for analysis were provided by Biolegend: anti-human PD-1-Brilliant Violet 421 (329920),CXCR3-APC (353708), CXCR5-PerCP-Cy5.5 (356910), CCR4-PE-Cy7 (359410), CD62L-PerCP/Cy5.5 (304824) and CD226-PerCP/Cy5.5 (338314). Monoclonal antibodies provided by ThermoFisher included TIGIT-PE (12–9500-42), CD4-PE-Cyanine7 (25–0048-42), CD4-eFluor450 (48-0048-42), CD4-APC (MHCD0405), 2B4-APC (17-5837-42), CD3-Alexa Fluor 700 (56-0032-82), CD3-FITC (11-0039-42), CD45RA-APC-eFluor780 (47-0458-42), CD25-PE-Cyanine7 (25-0259-42), CD127-APC-eFluor780 (47-1271-82), CD155-PE (12-1550-41), CD112-APC (17-1128-42), CD19-FITC (11-0199-42), rhAnnexin V-FITC (BMS147FI), 7-AAD Viability Staining Solution (00-6993-50), TIM-3-PerCP-eFluor 710 (46-3109-42) and CD8-Pacific Orange (MCD0830). For viability assays, rhAnnexin V/FITC, 7-AAD Viability Staining Solution was used. Data acquisition was performed on Gallios™ Flow Cytometer research system (Beckman Coulter) and data analysis was performed using Kaluza 1.2 Flow Cytometry Analysis Software (Beckman Coulter).

Catakovic K., Gassner F.J., Ratswohl C., Zaborsky N., Rebhandl S., Schubert M., Steiner M., Gutjahr J.C., Pleyer L., Egle A., Hartmann T.N., Greil R, & Geisberger R. (2017). TIGIT expressing CD4+T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia. Oncoimmunology, 7(1), e1371399.

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Rituximab Effects on T and B Cells in MS

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B and T cell numbers were analyzed before and after rituximab treatment in 179 MS patients (Table S1). Whole blood was collected, lysed with IOTest^® 3 Lysing Solution (Beckman Coulter) and stained with an antibody mix containing antibodies against CD19 and CD3 (multicolor panel from Beckman Coulter including antibodies against CD45, CD56, CD19 and CD3 and in addition an antibody against CD16 from BD). For the quantification of absolute T and B cell numbers Flow-Count Fluorospheres (Beckman Coulter) with a known concentration were added at an equal volume to the stained sample. Measurements were performed on a Navios (Beckman Coulter) flow cytometer until March 2016 and on an Aquios (Beckman Coulter) flow cytometer after April 2016. The number of cells was calculated as follows: absolute count (cells/μl) = (total number of cells counted / total number of fluorospheres counted) x concentration of Flow-Count Fluorospheres. T cell subtypes and activated T cells were analyzed in 9 RRMS samples before (untreated) and after rituximab therapy (Table S1) using a multicolor panel with appropriate antibodies (Key Resources Table). PBMCs from untreated RRMS (REM) patients used for the in vitro CFSE assay, were stained ex vivo for multiple B cell markers (Key Resources Table) to distinguish different B cell subsets and to correlate their frequency with the AP of T cells in vitro.

Jelcic I., Al Nimer F., Wang J., Lentsch V., Planas R., Jelcic I., Madjovski A., Ruhrmann S., Faigle W., Frauenknecht K., Pinilla C., Santos R., Hammer C., Ortiz Y., Opitz L., Grönlund H., Rogler G., Boyman O., Reynolds R., Lutterotti A., Khademi M., Olsson T., Piehl F., Sospedra M, & Martin R. (2018). Memory B Cells Activate Brain-Homing, Autoreactive CD4+ T Cells in Multiple Sclerosis. Cell, 175(1), 85-100.e23.

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Isolation and Culture of Dental Pulp Cells

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Normal human third molars were extracted from six patients (age, 18-29 years) at the Aichi-Gakuin University Dental Hospital after obtaining written, informed consent. The dental pulp of six teeth was gently removed, and DPCs were isolated as previously described (15) , with slight modification. Briefly, the harvested dental pulp tissue was minced into 1-3-mm 2 pieces and enzymatically digested. A total of 3 × 10 4 cells were isolated based on their ability to form cell colonies and seeded onto 35-mm dishes (BD Biosciences, San Jose, CA, USA) after filtration through a 40-µm cell strainer (BD Biosciences) and lysis of red blood cells with IOTest3 lysing solution (Beckman Coulter, Fullerton, CA, USA). Cells were detached and subcultured when they reached 70% confluence. At passage 6, DPCs had a flat, spreadout, and fibroblast-like morphology; these cells were used in subsequent experiments.

Okuwa Y., Toriumi T., Nakayama H., Ito T., Otake K., Kurita K., Nakashima M, & Honda M. (2018). Transplantation effects of dental pulp-derived cells on peripheral nerve regeneration in crushed sciatic nerve injury. Journal of oral science, 60(4).

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