Rbc lysing solution

Manufactured by BD

Sourced in United States

RBC Lysing Solution is a laboratory reagent used to lyse (break down) red blood cells (RBCs) in a sample. It is a key component in various hematological analyses and sample preparation procedures.

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Lysing solution (136 citations)

11 protocols using rbc lysing solution

Multicolor Flow Cytometry Analysis

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Premixed cocktail of monoclonal antibodies: CD3-PerCP, CD4-FITC, and CD8-PE (BD Biosciences, USA) were added into 100 µL of EDTA blood in a 12 mm × 75 mm tube. The tube was gently mixed and incubated at room temperature for 20 min in the dark, then added 2 mL of RBC lysing solution (BD Biosciences, USA) and incubated for another 10 min. Centrifugation at 500 g for 5 min and subsequently washed with 2 mL of PBS, the cell pellets were resuspended in 0.5 mL of 1% paraformaldehyde in PBS. The stained cells were then analyzed by a flow cytometer (FACSverse, BD Biosciences, USA), using CellQuest software program (BD Biosciences, USA). The percentage of CD3⁺, CD4⁺, and CD8⁺ lymphocytes and CD4⁺/CD8⁺ were determined from the CD3⁺/CD4⁺ and CD3⁺/CD8⁺ tubes, using FL1 and FL2 (31 ).

Mao X., Gu C., Ren M., Chen D., Yu B., He J., Yu J., Zheng P., Luo J., Luo Y., Wang J., Tian G, & Yang Q. (2018). l-Isoleucine Administration Alleviates Rotavirus Infection and Immune Response in the Weaned Piglet Model. Frontiers in Immunology, 9, 1654.

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Multiparameter B Cell Immunophenotyping

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One hundred μl of blood were stained for 20 min at room temperature with the following antibodies: anti-CD19 (BD 555415), anti-CD27 (BD 555441), anti-IgD (BD 555778) to measure naive (IgD+CD27−), IgM memory (IgD+CD27+), switched memory (IgD−CD27+), late/exhausted memory (IgD−CD27−) B cells. After staining, red blood cells were lyzed using the RBC Lysing Solution (BD 555899), according to the manufacturer’s instructions. Up to 10⁵ events in the lymphocyte gate were acquired on an LSR-Fortessa (BD) and analyzed using FACS Diva (BD) software. Single color controls were included in every experiment for compensation.

Diaz A., Romero M., Vazquez T., Lechner S., Blomberg B.B, & Frasca D. (2017). Metformin improves in vivo and in vitro B cell function in individuals with obesity and Type-2 Diabetes. Vaccine, 35(20), 2694-2700.

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Single Cell Isolation and Analysis

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Single cell suspensions were generated from the indicated organs (see above) and 1×10⁶ cells were incubated with the indicated mAb at 4°C for 20 min; then erythrocytes were lysed with RBC Lysing Solution (BD PharMingen). Cells were analyzed using a LSRII (BD) flow cytometer and FlowJo software, gated on viable leukocytes using the live/dead fixable near-IR dead cell stain kit from Invitrogen (Paisley, UK).

Sims S., Colston J., Emery V, & Klenerman P. (2014). CD73 Is Dispensable for the Regulation of Inflationary CD8+ T-Cells after Murine Cytomegalovirus Infection and Adenovirus Immunisation. PLoS ONE, 9(12), e114323.

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Porcine Lymphocyte Subset Analysis

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Routine blood examination included lymphocytes, neutrophils, intermediate cells, red blood cells, haematocrit, mean corpuscular volume, platelets, thrombocytocrit, and white blood cells. These parameters were analyzed using an automatic blood analyzer (Advia 120, Bayer HealthCare, Tarrytown, NY). Lymphocyte subtype was measured by a FACS Calibur flow cytometer (Becton, Dickinson and Company, San Jose, CA). Briefly, mouse anti-porcine CD3, mouse anti-porcine CD4, and mouse anti-porcine CD8 (Southern Biotechnology Associates, Birmingham, AL, USA) were added into 100 μL of blood in a 12 mm × 75 mm tube. The tube was gently mixed and incubated for 30 min in the dark at room temperature, then added 1 mL of RBC lysing solution (BD Biosciences, USA) and incubated for another 10 min. The cocktail was centrifuged at 500×g for 5 min, then re-suspended with PBS and detected by flow cytometer. The percentage of CD3⁺, CD4⁺, and CD8⁺ lymphocytes were determined by CellQuest software program (BD Biosciences, USA).

Peng X., Wang R., Hu L., Zhou Q., Liu Y., Yang M., Fang Z., Lin Y., Xu S., Feng B., Li J., Jiang X., Zhuo Y., Li H., Wu D, & Che L. (2019). Enterococcus faecium NCIMB 10415 administration improves the intestinal health and immunity in neonatal piglets infected by enterotoxigenic Escherichia coli K88. Journal of Animal Science and Biotechnology, 10, 72.

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Immunophenotyping of Bone Marrow Cells

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Samples (2 ml) of fresh bone marrow (BM) were obtained from the patients and healthy controls. After filtration, 300 μl BM samples were divided into one control and two test tubes. An antibody against monoclonal mouse IgG1-PE (cat no. 555749; 1:5) was added as a negative control and the mouse monoclonal antibodies CD114-PE (cat no., 554538; 1:5) and CD117-PE (cat no., 340529; 1:5) were added to each test tube, respectively. Mouse monoclonal antibodies CD34-PerCP (cat no., 340430; 1:5) and CD59-FITC (cat no., 555763; 1:5) were stained in all tubes as markers of stem cells and the PNH clone. All antibodies were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Following incubation at 4°C for 30 min, red blood cells (RBCs) in the sample were lysed with 2 ml RBC lysing solution (BD Biosciences). Next, the bone marrow hemopoietic cells were washed twice with phosphate-buffered saline (PBS) and analyzed using a BD FACSCalibur flow cytometer (BD Biosciences) with CellQuest software, version 3.1. At least 100,000 cells were acquired for each sample.

FU R., DING S.X., LIU Y., LI L.J., LIU H., WANG H.L., ZHANG T, & SHAO Z.H. (2016). Expression and function of hematopoiesis-stimulating factor receptors on the GPI− and GPI+ hematopoietic stem cells of patients with paroxysmal nocturnal hemoglobinuria/aplastic anemia syndrome. Experimental and Therapeutic Medicine, 11(5), 1668-1672.

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Obese Donor PBMC and SVF B-cell Profiling

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Peripheral Blood Mononuclear Cells (PBMC) and SVF from obese donors (10⁶ cells) were stained for 20 min at room temperature (RT) with the following antibodies: Live/Dead detection kit (InVitrogen1878898), anti-CD45 (Biolegend 368540) and anti-CD19 (BD 555415). To evaluate naïve and memory B cells, PBMC and SVF were stained with anti-CD19, anti-CD27 (BD 555441) and anti-IgD (BD 555778) to measure naive (IgD + CD27-) and all memory B cells that include the subsets of IgM memory (IgD + CD27+), switched memory (IgD-CD27+), and double negative memory (DN, IgD-CD27-) B cells. After staining, red blood cells were lyzed using the RBC Lysing Solution (BD 555899), according to the manufacturer’s instructions. Up to 10⁵ events in the lymphocyte gate were acquired on an LSR-Fortessa (BD) and analyzed using FlowJo 10.5.3 software. Single color controls were included in every experiment for compensation. Isotype controls were also used in every experiment to set up the gates.

Frasca D., Romero M., Diaz A., Garcia D., Thaller S, & Blomberg B.B. (2021). B Cells with a Senescent-Associated Secretory Phenotype Accumulate in the Adipose Tissue of Individuals with Obesity. International Journal of Molecular Sciences, 22(4), 1839.

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Flow Cytometric Enumeration of Tregs

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To identify the percentage of Tregs in peripheral circulation, the flow cytometric procedure of Fujimoto et al. was followed with slight modification.^{9 (link)} In brief, a 25 μl aliquot of whole blood was diluted with 75 μl of phosphate buffered saline (PBS, pH 7.4) and the diluted blood samples were incubated with 10 μl each of FITC anti-human CD4 and PE anti-human CD25 monoclonal antibodies (BioLegend, San Diego, CA, USA) and isotype-matched negative controls for 30 min in the dark at room temperature. The erythrocytes were then lysed by incubating the samples with 2 ml of RBC lysing solution (BD biosciences, CA, USA) for 5 min at room temperature. Thereafter, the cells were fixed with 0.5% paraformaldehyde and 15,000 events were acquired and analyzed in a flow cytometer (FACS Calibur with sorter, BD biosciences, San Jose, CA, USA). Cells were identified from their characteristic forward and side scatter profile on dot plots and gated. Data acquisition and analysis of FL-1 (FITC) and FL-2 (PE) were done using Cell Quest software (BD biosciences, USA). The relative proportion of Tregs was calculated from the statistical package of the Cell Quest software from quadrant gate setting.

Mondal N.K., Sobieski M.A., Pham S.M., Griffith B.P., Koenig S.C., Slaughter M.S, & Wu Z.J. (2017). Infection, Oxidative Stress and Changes in Circulating Regulatory T cells of Heart Failure Patients Supported by Continuous-Flow Ventricular Assist Devices. ASAIO journal (American Society for Artificial Internal Organs : 1992), 63(2), 128-133.

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Measurement of Switched Memory B Cells

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One hundred μl of blood were stained for 20 min rt with anti-CD19 (BD 555415), anti-CD27 (BD 555441), anti-IgD (BD 555778) to measure switched memory B cells (IgD-CD27+). After staining, red blood cells were lyzed using the RBC Lysing Solution (BD 555899). For intracellular staining of TNF-α, after membrane staining with anti-CD19, cells were fixed, permeabilized with 1X-PBS/0.2% Tween-20 and incubated with anti-TNF-α (BD 554512). Up to 10⁵ events in the lymphocyte gate were acquired on an LSR-Fortessa (BD) and analyzed using FACS Diva (BD) software. Single color controls were included in every experiment for compensation.

Frasca D., Diaz A., Romero M., Landin A.M, & Blomberg B.B. (2015). Cytomegalovirus (CMV) seropositivity decreases B cell responses to the influenza vaccine. Vaccine, 33(12), 1433-1439.

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Flow Cytometric Intracellular TNF-α Assay

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One hundred µl of blood were stained for 20 min at room temperature. Red blood cells were lysed using the RBC Lysing Solution (BD). For intracellular TNF-α, cells were fixed and permeabilized with 1X-PBS/0.2%Tween. Up to 10⁵ events in the lymphocyte gate were acquired on an LSR-Fortessa (BD) and analyzed using FACS Diva software.

Frasca D., Ferracci F., Diaz A., Romero M., Lechner S, & Blomberg B.B. (2016). Obesity decreases B cell responses in young and elderly individuals. Obesity (Silver Spring, Md.), 24(3), 615-625.

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Flow Cytometry Analysis of Lymphocyte Subsets

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Lymphocyte subtypes were analysed by a flow cytometer (FACSVerse, BD Biosciences, USA). A premixed cocktail of monoclonal antibodies (CD3-FITC, CD4-Percp and CD8-PE (BD Biosciences, USA)) were added into a 12 mm × 65 mm tube with 100 μL EDTA blood samples, and then the mixtures were gently mixed in the dark for 30 min. Thereafter, 2 mL RBC lysing solution (BD Biosciences, USA) was added to the tube and incubated for another 10 min. The mixture was centrifuged at 500 g for 5 min and washed with 2 mL of PBS. The cell pellets were resuspended in 1 mL of 1% paraformaldehyde in PBS and measured by a flow cytometer. The percentage of T-helper cells (CD3⁺CD4⁺) and cytotoxic T-cells (CD3⁺CD8⁺) were analysed by FlowJo software (FlowJo LLC, Ashland, OR, USA).

Tang W., Chen D., Yu B., He J., Huang Z., Zheng P., Mao X., Luo Y., Luo J., Wang Q., Wang H, & Yu J. (2020). Capsulized faecal microbiota transplantation ameliorates post-weaning diarrhoea by modulating the gut microbiota in piglets. Veterinary Research, 51, 55.

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