Rbc lysis buffer

Manufactured by BD

Sourced in United States, Canada

RBC lysis buffer is a laboratory reagent used to lyse or break down red blood cells (RBCs) in a biological sample. It disrupts the RBC cell membrane, releasing the cellular contents. This process is commonly used as a sample preparation step in various analytical techniques, such as flow cytometry, where the removal of RBCs is necessary to focus on other cell types of interest.

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Spelling variants of this product

Lysis buffer (118 citations) RBC lysis (4 citations)

94 protocols using rbc lysis buffer

Isolation of Peritoneal Macrophages

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Mice were intraperitoneally injected with 3 ml of 3% thioglycolate broth medium and sacrificed via cervical dislocation after 4 days. After being immersed in 75% alcohol for 10 min, the sacrificed mice were placed in a supine position on an ultra-clean workbench where their abdominal skins were cut open and then flushed twice with 5 ml of pre-chilled Roswell Park Memorial Institute (RPMI) 1640 medium (supplemented with 10 U/ml of penicillin and 100 μg/ml of streptomycin), followed by a collection of their peritoneal lavage fluid (PLF). After centrifugation (1,000 rpm, 5 min), the resulting supernatant of the PLF was discarded, and the pellet was resuspended with 5 ml of 1× RBC Lysis Buffer (555899, BD Biosciences, San Jose, CA, USA) and incubated in an ice bath for 10 min. After a second centrifugation step, the resulting supernatant was discarded again, and the cell pellet was resuspended and plated in RPMI 1640 medium containing 10% fetal bovine serum (FBS) for cultivation.

Shi X., Li T., Liu Y., Yin L., Xiao L., Fu L., Zhu Y., Chen H., Wang K., Xiao X., Zhang H., Tan S, & Tan S. (2022). HSF1 Protects Sepsis-Induced Acute Lung Injury by Inhibiting NLRP3 Inflammasome Activation. Frontiers in Immunology, 13, 781003.

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Neutrophil Quantification in Murine Bronchoalveolar Lavage

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BAL from healthy and infected mice were obtained as described above. After centrifugation, cell pellets were suspended with 1× RBC lysis buffer (BD Bioscience; CA, USA) and incubated at room temperature for 3 mins. Cells were washed with PBS twice followed by resuspension with 100μl 1× PBS. Cells were counted using an automated hematology analyzer. 1×10⁵ cells were then spun down using a cytospin machine (Thermo Scientific; MA, USA) for 8 mins at room temperature. Slides were allowed to air dry before H&E staining with Hemacolor staining kit (EMD Millipore; Germany) following manufacture’s protocol. After staining, slides were allowed to air dry overnight before mounting with Vectashield (Vector Laboratories; CA, USA) and coverslip. Slides were then analyzed with a microscope and neutrophils were identified and counted based on their nuclear appearance.

Wong C.K., Smith C.A., Sakamoto K., Kaminski N., Koff J.L, & Goldstein D.R. (2017). Aging impairs alveolar macrophage phagocytosis and increases influenza-induced mortality in mice. Journal of immunology (Baltimore, Md. : 1950), 199(3), 1060-1068.

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Isolation and Sorting of Monocytes

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PBMC isolated from non-BCG vaccinated healthy volunteers as described above were treated with red blood cell (RBC) lysis buffer (Becton Dickinson, USA). Cells were washed, filtered and counted and extracellular staining was performed with mouse anti-human monoclonal CD3-PerCPCy5.5 (eBioscience, San Diego, USA) and mouse anti-human CD14-Alexa Fluor 488 (Becton Dickinson, USA) in PBS supplemented with 2% FBS and EDTA 0.8 mM for 25 min at +4⋅C. The cells were processed through either BC Moflo XDP (Beckman Coulter, California) or BD FACSAriaIII (Becton Dickinson, San Jose, California) high speed cell sorters at Flow Cytometry Core Facility at the Institute of Child Health, University College London. Sorting the monocyte population from one sample required at least 3 h using any of the two cell sorters and therefore in order to process samples from two healthy volunteers during every experiment, we used concurrently both cell sorters. Monocytes were considered the cells which were CD3⁻CD14⁺. Single colored-stained CD3⁺ and CD14⁺ cells were used as controls for gating the CD3⁻CD14⁺ monocytes. Single stained CD3⁺ and CD14⁺ CompBeads (Becton Dickinson, USA) were used as compensation controls.

Polycarpou A., Holland M.J., Karageorgiou I., Eddaoudi A., Walker S.L., Willcocks S, & Lockwood D.N. (2016). Mycobacterium leprae Activates Toll-Like Receptor-4 Signaling and Expression on Macrophages Depending on Previous Bacillus Calmette-Guerin Vaccination. Frontiers in Cellular and Infection Microbiology, 6, 72.

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Macrophage Immunophenotyping by Flow Cytometry

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Peritoneal lavage fluid was stained with F4/80-PE/Cy7 (BM8) (Biolegend) and CD11b-APC/A700 (M1I70) (eBioscience) antibodies for 35 min. Red blood cells (RBCs) were lysed with RBC lysis buffer (Becton Dickinson) for 5 min. Cells were fixed with 1% paraformaldehyde and analysed on an LSRII flow cytometer (BD). Macrophages were defined as F4/80+CD11b+ cells.

Band V.I., Crispell E.K., Napier B.A., Herrera C.M., Tharp G.K., Vavikolanu K., Pohl J., Read T.D., Bosinger S.E., Trent M.S., Burd E.M, & Weiss D.S. (2016). Antibiotic failure mediated by a resistant subpopulation in Enterobacter cloacae. Nature microbiology, 1(6), 16053.

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Intracellular Cytokine Staining in WBA

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To determine the cellular source/s of cytokines in the WBA, the cultures were stimulated for 16–24 hours. To block cytokine secretion cultures were for the last 6–8 hours of stimulation treated with GolgiStop (BD Biosciences) according to manufactures instructions. Following lysis of RBC using BD RBC lysis buffer (BD Biosciences), cells were surface stained using combinations of FITC, PE and PerCP/PE-Cy5 conjugated antibodies directed to CD3 (Clone UCHT1), CD4 or CD8 (all from BD Biosciences). Surface stained cells were fixed and permeabilized using BD Cytofix/Cytoperm, as per manufactures instruction, washed in permeabilization buffer (BD) and stained for presence of intracellular IFNγ and IL-10 using APC and PE conjugated antibodies (both from Pharmingen) respectively. Following intra cellular staining (ICS), samples were acquired on FACSort (BD Biosciences) and analyzed using CellQuest Pro (BD) or FlowJo (Treestar) software. Analysis was done on cells gated as viable lymphocytes based on their forward–side scatter. SEB (10 µg/ml) stimulated samples were used as positive control for ICS (not shown).

Kumar R., Singh N., Gautam S., Singh O.P., Gidwani K., Rai M., Sacks D., Sundar S, & Nylén S. (2014). Leishmania Specific CD4 T Cells Release IFNγ That Limits Parasite Replication in Patients with Visceral Leishmaniasis. PLoS Neglected Tropical Diseases, 8(10), e3198.

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Evaluating Microglial and Macrophage Interactions with γδ T Cells

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On day 3 after I/R, spleens from vehicle-treated rats were isolated and single splenocyte suspension was prepared by mechanical dissociation in 40-µm cell strainers. Red blood cells were lysed with BD RBC lysis buffer. Splenocytes were incubated with FITC anti-TCR_γδ Ab for 15 min on ice. TCR_γδ⁺ cells were sorted by flow cytometry. CD45^hi CD11b/c⁺ macrophages and CD45^lo CD11b/c⁺ microglia were sorted from vehicle-treated and rapamycin-treated ischemic brain hemispheres by flow cytometry, respectively. All cells were resuspended in supplemented RPMI 1640. 2 × 10⁵ macropahges or microglia were seeded into each well of a 96-well transwell plate (Corning). 1.25 × 10⁴ γδ T cells were seeded into each well of the insert. The cells were cultured at 37°C for 18 h. All cells in the lower wells (not the insert wells) were then incubated in 1 mM EDTA-PBS for 10 min and collected. Cells were incubated with PE anti-CD3 Ab for 15 min on ice. The number of CD3⁺ cells was enumerated by flow cytometry.

Xie L., Sun F., Wang J., Mao X., Xie L., Yang S.H., Su D., Simpkins J.W., Greenberg D.A, & Jin K. (2014). mTOR signaling inhibition modulates macrophages/microglia-mediated neuroinflammation and secondary injury via regulatory T cells after focal ischemia. Journal of immunology (Baltimore, Md. : 1950), 192(12), 6009-6019.

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Isolation and Purification of Splenocytes

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Spleens were removed and cut into pieces in cold PBS. After filtration through a 200-gauge steel mesh and removal of red blood cells with an RBC lysis buffer (BD Biosciences, USA), splenocytes were collected for further experimentation. Preparation of LPLs was performed as previously described (Weigmann et al. 2007 (link)).

Ma Y., Xu X., Li M., Cai J., Wei Q, & Niu H. (2019). Gut microbiota promote the inflammatory response in the pathogenesis of systemic lupus erythematosus. Molecular Medicine, 25, 35.

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Neutrophil Isolation from Peripheral Blood

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Neutrophils were separated from 3 ml peripheral blood samples using Ficoll‐Hypaque density gradient centrifugation, according to the manufacturer’s instructions (GE Healthcare). The purity of neutrophils was within acceptable parameters (higher than 95%). Peripheral blood samples (1 ml) were lysed twice with RBC lysis buffer (BD Biosciences) to obtain leukocytes.

Niu X., Yin L., Yang X., Yang Y., Gu Y., Sun Y., Yang M., Wang Y., Zhang Q, & Ji H. (2022). Serum amyloid A 1 induces suppressive neutrophils through the Toll‐like receptor 2–mediated signaling pathway to promote progression of breast cancer. Cancer Science, 113(4), 1140-1153.

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Orthotopic Pancreatic Tumor Xenograft Model

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Mice were anaesthetized using continuous-flow isoflurane, and their abdomen was sterilized. An incision was then made over the left upper quadrant of the abdomen, and the pancreas was exteriorized onto a sterile field. 1.0x10⁴ tumor cells in 50 μl sterile DMEM were injected into the tail of the pancreas via a 27 5/8” gauge needle. Successful injection was confirmed by the formation of a liquid bleb at the site of injection with minimal fluid leakage. The pancreas was then gently placed back into the peritoneal cavity, and the peritoneum and overlying skin were closed with 4-0 coated Vicryl violet FS-2 sutures (Ethicon). Tumors, lungs, and livers were harvested, weighed, and measured 4-6 weeks following implantation. 200 μl of blood was drawn from tumor-bearing animals via cardiac puncture with a 1 mL insulin syringe coated with 0.5M EDTA pH 8.0 (Gibco) to prevent coagulation and was immediately placed in a 150 mm gridded plate containing RBC lysis buffer (BD Biosciences). After 10 min. of lysis at room temperature, PBS was added to the plate and CTCs were directly visualized on a fluorescent microscope and counted.

Yuan S., Natesan R., Sanchez-Rivera F.J., Li J., Bhanu N.V., Yamazoe T., Lin J.H., Merrell A.J., Sela Y., Thomas S.K., Jiang Y., Plesset J.B., Miller E.M., Shi J., Garcia B.A., Lowe S.W., Asangani I.A, & Stanger B.Z. (2020). Global regulation of the histone mark H3K36me2 underlies epithelial plasticity and metastatic progression. Cancer discovery, 10(6), 854-871.

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Immune Cell Phenotyping Protocol

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A total of 300 μL PB was collected in a tube, and 5 μL per sample was incubated with each antibody except Foxp3-Alexaflour 647 for 20 min at room temperature in the dark. Erythrocytes were lysed using RBC Lysis Buffer (BD, Biosciences, USA) for 10 min in the dark. After completely washing the cells, they were fixed and permeabilized with the Foxp3 Fix/Perm Buffer Set (Biolegend, San Diego, USA) according to the manufacturer's instructions. Following incubation with the Foxp3 mAb for 30 min, cells were washed and resuspended with 1X Flow Cytometry Perm Buffer for analysis by flow cytometry. All samples were analyzed with a BD Verse flow cytometer (BD, Biosciences, USA), and FlowJo software was used to analyze data.

Jin Z., Ye W., Lan T., Zhao Y., Liu X., Chen J., Lai J., Chen S., Zhong X, & Wu X. (2020). Characteristic of TIGIT and DNAM-1 Expression on Foxp3+ γδ T Cells in AML Patients. BioMed Research International, 2020, 4612952.

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