Versalyse lysing solution

Manufactured by Beckman Coulter

Sourced in United States, France

VersaLyse Lysing Solution is a reagent designed for the lysis of red blood cells in whole blood samples. It facilitates the preparation of samples for flow cytometry analysis by selectively lysing erythrocytes, leaving the leukocyte population intact for further investigation.

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32 protocols using versalyse lysing solution

Flow Cytometric Analysis of Immune Cells

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Rats were dissected under anesthesia at the end of all exposures. Blood samples were collected, and splenocytes and thymocytes were extracted and analyzed using flow cytometry. Blood specimens were mixed with one-fifth volumes of 50-mM ethylenediaminetetraacetic acid (EDTA) solution, and 75-µl blood–EDTA mixtures were hemolyzed using 10-fold volumes of VersaLyse Lysing Solution (Beckman Coulter Inc., Brea, CA, USA), followed by incubation at room temperature for 10 min. Cells were washed twice in phosphate-buffered saline (PBS) and collected by centrifugation. Splenocytes and thymocytes were prepared in Hank's Balanced Salt Solution (HBSS; Thermo Fisher Scientific Inc., Waltham, MA, USA) by mincing spleen or thymus tissues between frosted edges of glass slides (Matsunami Glass Ind. Ltd, Osaka, Japan). Cells were filtered using nylon mesh sheets and washed with HBSS. Both cell types were collected by centrifugation and hemolyzed using VersaLyse Lysing Solution. After resuspension in 100 µl of PBS, cells were stained with labeled antibodies and were analyzed using flow cytometry (see below).
To obtain RNA samples, spleen and thymus tissues were removed from control and exposed rats, later soaked in RNA (Qiagen, Valencia, CA, USA), kept at 4°C for 2–5 days, and then at −80°C until RNA extraction.

Ohtani S., Ushiyama A., Maeda M., Ogasawara Y., Wang J., Kunugita N, & Ishii K. (2015). The effects of radio-frequency electromagnetic fields on T cell function during development. Journal of Radiation Research, 56(3), 467-474.

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Immunophenotyping of NSCLC Peripheral Blood

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We conducted flow cytometry staining to investigate immune cell subsets in the peripheral venous blood of NSCLC patients. Initially, 50 μl of peripheral blood were used per staining panel employing the following monoclonal antibodies: CD45 KrO, CD3 PB, CD8 APC-Alexa Fluor 700, CD4 PB, HLA-DR APC, CD127 FITC, CD25 ECP, CD19 ECD, CD27 PB, IgD FITC, CD5 PC5.5, CD3 APC-Alexa Fluor 750, CD56 PE, and CD16 PE (Supplementary Tables 2, 3). Samples were briefly vortexed and incubated for 15 min in the dark at room temperature (RT). Afterward, 500 µl of VersaLyse Lysing Solution (Beckman Coulter Brea, California, USA) was added and samples were further vortexed and incubated for 20 min in the dark at RT. After that, samples were immediately analyzed by Navios EX Flow Cytometer (Backman Coulter) with subsequent analysis with Kaluza Analysis 2.1 software (Beckman Coulter). The panels were validated using the Fluorescence minus one (FMO) procedure.

Krizova L., Benesova I., Zemanova P., Spacek J., Strizova Z., Humlova Z., Mikulova V., Petruzelka L, & Vocka M. (2024). Immunophenotyping of peripheral blood in NSCLC patients discriminates responders to immune checkpoint inhibitors. Journal of Cancer Research and Clinical Oncology, 150(2), 99.

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Multiparametric flow cytometry analysis of immune cell subsets

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Blood samples (100 μL) were vortexed for 5 min and incubated for 15 min in the dark with 1.5 ml of VersaLyse Lysing Solution (Beckman Coulter, BC) to lyse red blood cells and select the total leukocyte population. Samples were incubated with antibodies of interest for 15 min in the dark: anti-human CD38-ECD, CD4-APC, CD8-BV605, CD3-BV510 (Biolegend). The stained cells were then washed with Dulbecco’s phosphate buffered saline, permeabilized with the IntraPrep Permeabilization Reagent kit and stained with anti-human IL-6 FITC (Immunotool), anti IFN-γ BV650 (BD Bioscences) and anti-IL-10 AF700 (BC). For certain analyses, the DuraClone IM T-cell subset tube (B53328, BC) was used and gated according to the Manufacturer’ instructions.
All stained cells were analyzed using CytoFLEX software (Beckman Coulter) and CytExpert 2.3 (BC). Results were expressed as percentage of positive cells.

Minutolo A., Petrone V., Fanelli M., Maracchioni C., Giudice M., Teti E., Coppola L., Sorace C., Iannetta M., Tony Miele M., Bernardini S., Mastino A., Sinibaldi Vallebona P., Balestrieri E., Andreoni M., Sarmati L., Grelli S., Garaci E, & Matteucci C. (2023). Thymosin alpha 1 restores the immune homeostasis in lymphocytes during Post-Acute sequelae of SARS-CoV-2 infection. International Immunopharmacology, 118, 110055.

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Isolation of Stem-like CTC Subpopulations

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Stem EpCAM-negative CTCs (CTC-5 (EpCAM-CD45-CD44+CD24-Ncadh-) and CTC-6 (EpCAM-CD45-CD44+CD24-Ncadh+) subpopulations) were sorted using a cell sorter MoFlo XDP with Summit software (Beckman Coulter, USA). For this procedure, stabilized heparin venous blood was incubated with fluorochrome-labeled monoclonal antibodies to CD45 (PE/Cy7, mouse IgG1, clone HI30, BD Pharmingen, San Diego, CA, USA), CD44 (APC-H7, mouse IgG2b, clone G44-26, BD Pharmingen, USA), CD24 (PerCP-Cy5.5, mouse IgG2a, clone ML5, BD Pharmingen, USA), EpCAM (FITC, mouse IgG1, clone EBA-1, BD Pharmingen, USA) and CD325 (N-Cadherin) (PE, mouse IgG1, clone 8C11, BD Pharmingen, USA). Isotype control antibodies (mouse PE-Cy7 IgG1, mouse APC-H7 IgG2b, mouse PerCP-Cy5.5 IgG2a, mouse PE IgG1, BD Pharmingen, USA) were used for negative control. Then erythrocytes were lysed in 1 mL VersaLyse Lysing Solution (Beckman Coulter, USA). Side scatter and forward scatter profiles were used to eliminate cell doublets. Then EpCAM-CD45-CD44+CD24-Ncadh- and EpCAM-CD45-CD44+CD24-Ncadh+ cells were routinely sorted.

Kaigorodova E.V., Savelieva O.E., Tashireva L.A., Tarabanovskaya N.A., Simolina E.I., Denisov E.V., Slonimskaya E.M., Choynzonov E.L, & Perelmuter V.M. (2018). Heterogeneity of Circulating Tumor Cells in Neoadjuvant Chemotherapy of Breast Cancer. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(4), 727.

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Brain Metastasis Mouse Model with Adoptive Transfer

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The brain metastasis model was prepared as described previously [9 (link)]. In brief, SCID mice were anesthetized by isoflurane and a microclamp was applied to the external carotid artery. Then Nluc-H1915 cells (1 × 10⁵ cells/head) were injected slowly into the internal carotid artery by surgical visualization, and finally the cut made in the skin was stitched up. Twenty-four days after tumor inoculation, blood was collected from the jugular vein and Nluc activity in plasma was measured and used to randomize mice into control and test groups (day 1). In parallel, spleens were harvested from BALB/c mice, which were subcutaneously immunized with H1915 cells one week before, and splenocytes were prepared with Hank’s balanced salt solution (Sigma-Aldrich) after treatment with VersaLyse lysing solution (Beckman Coulter, Brea, CA, USA). This splenocytes (5 × 10⁴ cells /head) were intravenously injected into the tail of brain metastasis model mice as donor lymphocyte infusion (DLI) on day 1 and mouse IgG and anti-PD-L1 antibody were administered intraperitoneally at a dose of 10 mg/kg twice a week.

Masuda C., Morinaga M., Wakita D., Yorozu K., Kurasawa M., Sugimoto M, & Kondoh O. (2021). PD-L1 blockade exhibits anti-tumor effect on brain metastasis by activating CD8+ T cells in hematogenous metastasis model with lymphocyte infusion. Clinical & Experimental Metastasis, 39(2), 335-344.

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Comprehensive Immunophenotyping of B Cells

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B cell whole peripheral blood samples (200 μL) were stained with the following anti-human monoclonal antibodies IgD-Alexa Fluor 488 (cat. 348216, BioLegend, Inc., San Diego, CA, USA), CD38-PE (cat. A07779, Beckman Coulter, Brea, CA, USA), CD5-ECD (cat. A33096, Beckman Coulter, USA), CD27-PC7 (cat. A54823, Beckman Coulter, Brea, CA, USA), CD19-APC/Cy7 (cat. 302218, BioLegend, Inc., USA), and CD45-Krome Orange (cat. A96416, Beckman Coulter, USA), all antibodies were utilized at the dilutions that were recommended by the manufacturers. After incubation at room temperature in the dark for 10 min, erythrocytes were lysed for 15 min by adding 2 mL of VersaLyse Lysing Solution (Beckman Coulter, Inc., USA) supplied with 50 μL IOTest 3 Fixative Solution (Beckman Coulter, Inc., USA). Next, cells were washed (7 min, 330 g) twice with a buffer (sterile phosphate-buffered saline (PBS) containing 2% of heat inactivated fetal bovine serum, Sigma-Aldrich, St. Louis, MO, USA) and were resuspended in 0.5 mL PBS containing 2% of neutral buffered formalin solution (Sigma-Aldrich, St. Louis, MO, USA). Sample acquisition was performed using a Navios flow cytometer (Beckman Coulter, Inc., USA), equipped with 405, 488 and 638 nm lasers. There were collected at least 5000 CD19+ B cells to be analyzed in each sample.

Kudryavtsev I.V., Arsentieva N.A., Batsunov O.K., Korobova Z.R., Khamitova I.V., Isakov D.V., Kuznetsova R.N., Rubinstein A.A., Stanevich O.V., Lebedeva A.A., Vorobyov E.A., Vorobyova S.V., Kulikov A.N., Sharapova M.A., Pevtcov D.E, & Totolian A.A. (2021). Alterations in B Cell and Follicular T-Helper Cell Subsets in Patients with Acute COVID-19 and COVID-19 Convalescents. Current Issues in Molecular Biology, 44(1), 194-205.

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Neutrophil Immunophenotyping and Cytokine Profiling

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Whole blood was incubated with the monoclonal antibodies anti-CD15 at the fluorochrome fluorescein isothiocyanate (FITC, emission 525 nm, Immunotech, Marseille, France), anti-CD64 at the phycoerythrin (PE, emission 575 nm CYTO-STAT, Miami, Florida, US), and anti-CD45 at the fluorochrome phycoerythrin-Cy5 (PC5, emission 650 nm, Immunotech). Red blood cells were lysed using VersaLyse Lysing Solution (Beckman Coulter, Immunotech, Marseille, France). White blood cells were analyzed after running through the Cytomics FC-500 flow cytometer with gating for granulocytes based on their characteristic SS/CD45 expression. Granulocytes expressing the surface marker CD15 were considered neutrophils. Fluorospheres (Immunotech) were used for the determination of absolute counts. IgG isotypic controls at the fluorochromes FITC and PE (Immunotech) were analyzed before the start of the analysis for every patient.
Serum was collected from each patient and stored at -70 °C until assay. Concentrations of interleukin-8 (IL-8) and IFNγ were measured in duplicate by enzyme immunoassay (Affymetrix, Santa Clara, CA, USA). The lower detection limits was 7.8 pg/ml for IL-8 and 19.5 pg/ml for IFNγ.

Xini A., Pistiki A., Lada M., Giamarellos-Bourboulis E.J, & Dimopoulos G. (2019). Association of the early absolute CD64-expressing neutrophil count and sepsis outcome. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology, 38(6).

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Multiparameter Flow Cytometry Panels

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Fresh whole blood (100 µL) was labelled for surface and intracellular markers distributed in 3 flow cytometry panels: panel 1: CD3, CD8, CD4, CD45RA, CD27, CD28 and CD95, panel 2: CD3, CD8, CD4, CD45RA, CD27, CD28, Ki-67 and Sestrin-2 and panel 3: CD3, CD8, CD4, CD56, CD57, NKG2A, KLRG1 and DAP12 (see Table S4 for the specific clones and brand). All were direct stainings except for Sestrin-2. All stainings were performed according to the manufacturer’s recommendations, with Sestrin-2 expression being assessed after fixation and permeabilization (Biolegend FoxP3 Perm and Fix). After labelling, red blood cells were lysed using Versalyse lysing solution (Beckman Coulter, Inc., USA) according to the manufacturer’s recommendations. Surface and intracellular markers were analysed by flow cytometry using a Navios® flow cytometer (Beckman Coulter, Inc., USA) according to the manufacturer’s recommendations. Flow set and Flow-check fluorosphere (Beckman Coulter, Inc., USA) were used to calibrate our cytometer on days of experiment. Fluorescence minus one (FMO) controls were used to verify the absence of spillover after applying the compensation matrix and as gating controls. Additionally, isotype controls were used for Sestrin-2, Dap-12, Ki-67, NKG2A and CD95.

Poisson J., El-Sissy C., Serret-Larmande A., Smith N., Lebraud M., Augy J.L., Conti C., Gonnin C., Planquette B., Arlet J.B., Hermann B., Charbit B., Pastre J., Devaux F., Ladavière C., Lim L., Ober P., Cannovas J., Biard L., Gulczynski M.C., Blumenthal N., Péré H., Knosp C., Gey A., Benhamouda N., Murris J., Veyer D., Tartour E., Diehl J.L., Duffy D., Paillaud E, & Granier C. (2024). Increased levels of GM-CSF and CXCL10 and low CD8+ memory stem T Cell count are markers of immunosenescence and severe COVID-19 in older people. Immunity & Ageing : I & A, 21, 28.

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HERV-W ENV Expression in COVID-19

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Blood cells (100 μL) were incubated for 15 min in the dark with VersaLyse Lysing Solution (Beckman Coulter, BC) to lyse red blood cells and then permeabilized with IntraPrep Permeabilization Reagent kit (BC), followed by staining with anti HERV-W ENV monoclonal antibody (ENV-W01; GeNeuro) and a secondary FITC-labeled goat anti-mouse antibody (Beckton Dickinson, BD). For the analysis of leukocyte subpopulations, before intracellular staining, the cells were incubated with monoclonal antibodies of interest: CD4-APC and CD56-PE (BD), CD8-APC700, CD3-APC750, CD14-PE, CD19 ECD or PC7 (BC). For certain analyses, the DuraClone IM T-cell subsets Tube (B53328, BC) was used. Stained cells were then washed with PBS analysed via CytoFLEX and CytExpert 2.2 software (BC), at least 4.5 × 10⁵ cells in the gate of Leukocytes were recorded. The gating strategy is reported in Figure S1. Results are expressed as the percentage of HERV-W ENV-positive cells in COV with respect to HD used as a control group.

Balestrieri E., Minutolo A., Petrone V., Fanelli M., Iannetta M., Malagnino V., Zordan M., Vitale P., Charvet B., Horvat B., Bernardini S., Garaci E., di Francesco P., Sinibaldi Vallebona P., Sarmati L., Grelli S., Andreoni M., Perron H, & Matteucci C. (2021). Evidence of the pathogenic HERV-W envelope expression in T lymphocytes in association with the respiratory outcome of COVID-19 patients. EBioMedicine, 66, 103341.

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Comprehensive Immunophenotyping of Leukemia

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Immunophenotyping of leukemia patients was performed by flow cytometry. BM and/or PB samples were incubated with VersaLyse Lysing Solution (A09777, Beckman Coulter, USA) at 37°C for 2 minutes. Then, leukemic cells were isolated by centrifugation at 400×g for 3 minutes and 30 seconds. The supernatant was removed, and the cells were washed and fixed with IOTest 3 Fixative Solution (A07800, Beckman Coulter) and incubated with monoclonal antibodies. The resulting samples were assayed using a Cytomics FC500 (Beckman Coulter) flow cytometer and CXP Software. At least 3 × 10⁶ cells were analyzed from each sample.
The expression of CD2, CD3, cCD3, CD4, CD5, CD7, CD10, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, sCD22, CD23, CD25, CD26, CD30, CD33, CD34, CD38, CD43, CD45, CD56, CD61, CD64, CD65, CD71, cCD79a, CD103, CD117, CD138, HLA‐DR, TCR, FMC7, TdT, cMPO, Ki‐67, bcl‐2, and ZAP‐70 were analyzed.

Kolesnikova M.A., Sen'kova A.V., Pospelova T.I, & Zenkova M.A. (2021). Drug responsiveness of leukemic cells detected in vitro at diagnosis correlates with therapy response and survival in patients with acute myeloid leukemia. Cancer Reports, 4(4), e1362.

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