Rbc lysis buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, United Kingdom

RBC lysis buffer is a solution used to lyse (break down) red blood cells (RBCs) in a sample. It is commonly used in various applications, such as flow cytometry and cell isolation, to remove RBCs and enrich other cell types for analysis or further processing.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (30 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (29 mentions) Dnase 1, by Merck Group (28 mentions) Dnase 1, by Roche (28 mentions) Collagenase d, by Roche (20 mentions) Gentlemacs dissociator, by Miltenyi Biotec (17 mentions) Rpmi 1640, by Thermo Fisher Scientific (14 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (14 mentions) Percoll, by GE Healthcare (14 mentions) L glutamine, by Thermo Fisher Scientific (14 mentions) Ionomycin, by Merck Group (12 mentions) Ficoll paque plus, by GE Healthcare (11 mentions) Facsaria, by BD (11 mentions) 70 μm cell strainer, by BD (11 mentions) Collagenase 4, by Merck Group (10 mentions) Phorbol myristate acetate (pma), by Merck Group (10 mentions) Lsrfortessa, by BD (10 mentions) Sodium pyruvate, by Thermo Fisher Scientific (10 mentions) Hepes, by Thermo Fisher Scientific (10 mentions) Lsrii flow cytometer, by BD (9 mentions)

Close spelling variants of this product

RBC Lysis Buffer Solution EBioscience RBC lysis buffer EBioscience™ 1X RBC Lysis Buffer 10× RBC lysis buffer multispecies ACK RBC lysis buffer Multi-species RBC lysis buffer RBC lysis Lysis buffer

657 protocols using rbc lysis buffer

Isolation and Characterization of Immune Cells from RCC

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Peripheral blood samples were collected before surgery, and preserved in heparinized tubes at 4 °C until experimentation (within 2 h). After adding RBC Lysis Buffer.
(Thermo Fisher Scientific), white blood cells were extracted. Surgically-resected RCC samples were freshly minced and digested with collagenase IV (Sigma) and DNase I (Sigma) at 37 °C, strained through a 70-µm strainer, and then treated with RBC Lysis Buffer (Thermo Fisher Scientific). After Fc receptors blockade, peripheral white blood cells, or single cell suspensions, were stained at 4 °C with fluorescently labeled membrane marker antibodies for 30 min. Intracellular proteins were stained with appropriate antibodies after being dissolved in Intracellular Fixation & Permeabilization Buffer (Thermo Fisher Scientific). Peripheral white blood cells, or single cell suspensions, were stained with antibodies labeled with fluorochrome and maintained with cell staining buffer. Flowjo v10.0 was used for analyzing BD LSRFortessaTM X-20 (BD Biosciences) FACS data (Tree Star). Supplementary Table S3 provides information about antibodies in detail.

Wang J., Zhang S., Wang Y., Zhu Y., Xu X, & Guo J. (2024). RUNX3 pathway signature predicts clinical benefits of immune checkpoint inhibition plus tyrosine kinase inhibition in advanced renal cell carcinoma. BMC Urology, 24, 8.

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Tissue Dissociation and Leukocyte Isolation

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Cell suspensions were prepared from tissues by mechanical dissociation, followed by digestion in 5 mL of RPMI-1640 containing collagenase I (500 U/mL) (#SCR103; Sigma-Aldrich) and DNase I (0.2 mg/mL) (#04716728001; Sigma-Aldrich) for 45 min at 37°C on a shaker (220 rpm), followed by filtration through a 70-μm strainer and 25% Percoll (#GE17-0891-01; Sigma-Aldrich) gradient enrichment of leukocytes, and red blood cell (RBC) lysis. Tumor cells were recovered without Percoll enrichment. Blood cells were lysed in 5 mL of RBC lysis buffer (#A1049201; Thermo Fisher) three times for 5 min, and spleens were strained through a 70-μm filter in RPMI-1640 before lysing erythrocytes with RBC lysis buffer for 5 min. Single cells were restimulated and stained for surface and intracellular markers (see flow cytometry below).

Ferrer M., Mourikis N., Davidson E.E., Kleeman S.O., Zaccaria M., Habel J., Rubino R., Flint T.R., Connell C.M., Lukey M.J., White E.P., Coll A.P., Venkitaraman A.R, & Janowitz T. (2023). Ketogenic diet promotes tumor ferroptosis but induces relative corticosterone deficiency that accelerates cachexia. bioRxiv.

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Isolation and Characterization of Immune Cells from RCC Tissue

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Peripheral blood samples were collected before surgery, stored in heparinized tubes at 4 °C within two hours before the experiment. RBC Lysis Buffer (Thermo Fisher Scientific) was used to isolate white blood cells. RCC samples were collected and minced after resection. After digestion by collagenase IV (Sigma) and DNase I (Sigma) at 37 °C and passing through a 70-μm strainer, RCC samples were then treated with RBC Lysis Buffer (Thermo Fisher Scientific) to obtain single cell suspensions. Samples were then treated with Fc receptors blockade, and stained for 30 min at 4 °C with fluorescently labeled membrane marker antibodies. Intracellular proteins were stained with corresponding antibodies after being disposed by Intracellular Fixation and Permeabilization Buffer (Thermo Fisher Scientific). Samples were then stained by the fluorochrome-labeled antibodies and preserved in cell staining buffer. Flow-cytometry analysis were performed by BD LSRFortessa™ X-20 (BD Biosciences) and analyzed by Flowjo v10.0 (Tree Star). Antibodies used for flow cytometry are listed in Supplementary Table S4.

Wang J., Lin J., Wang J., Wang Y., Zhu Y., Xu X, & Guo J. (2024). Effect of Annexin A2 on prognosis and sensitivity to immune checkpoint plus tyrosine kinase inhibition in metastatic renal cell carcinoma. Discover. Oncology, 15, 86.

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Isolation and Analysis of Immune Cells

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Whole blood samples were analyzed for complete blood counts and leukocyte differentials utilizing the Advia 120 Hematology Analyzer (Siemens Healthcare Diagnostics). For the isolation of PBMCs, human blood samples underwent centrifugation at 282g for 10 minutes. Subsequently, supernatants were removed for plasma preparation as previously described (73 (link)), and white cell pellets were subjected to density gradient centrifugation using Ficoll (GE Healthcare) to isolate PBMCs.
Mouse white blood cells were prepared from whole blood by lysing RBCs with RBC Lysis Buffer (Thermo Fisher Scientific). Plasma was obtained from the whole blood supernatant through centrifugation at 500g for 10 minutes. Mouse liver single-cell suspensions were generated using a liver dissociation kit (Miltenyi Biotec) in combination with the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) according to the manufacturer’s guidelines. RBCs within the organ single-cell suspensions were lysed using RBC Lysis Buffer (Thermo Fisher Scientific).

Liu Y., Su S., Shayo S., Bao W., Pal M., Dou K., Shi P.A., Aygun B., Campbell-Lee S., Lobo C.A., Mendelson A., An X., Manwani D., Zhong H, & Yazdanbakhsh K. (2023). Hemolysis dictates monocyte differentiation via two distinct pathways in sickle cell disease vaso-occlusion. The Journal of Clinical Investigation, 133(18), e172087.

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Workflow for Single-Cell Analysis of Tumor Samples

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Before surgery, peripheral blood samples were collected from venous blood and preserved in heparin anticoagulant tubes at 4°C until experimentation (within 2 h). White blood cells were extracted by RBC Lysis Buffer (Thermo Fisher Scientific). RCC samples were obtained and examined just after surgical resection. At 37°C, freshly minced tumor samples were digested with collagenase IV (Sigma) and DNase I (Sigma), and then strained through a 70 μm strainer. The samples were then treated in RBC Lysis Buffer (Thermo Fisher Scientific). After blocking Fc receptors, single‐cell suspensions and white blood cells were stained separately for 30 min at 4°C with fluorescently labeled membrane marker antibodies. Intracellular proteins were stained with appropriate antibodies after being dissolved in Intracellular Fixation & Permeabilization Buffer (Thermo Fisher Scientific) according to the manufacturer's instructions. White blood cells and cell suspensions were stained with antibodies labeled with fluorochrome and maintained with cell staining buffer. Flowjo v10.0 was used to analyze BD LSRFortessaTM X‐20 (BD Biosciences) FACS data (Tree Star). Antibodies in detail are described in a previous publication.²⁶

Xu X., Zhang S., Wang Y., Zhu Y., Wang J, & Guo J. (2023). HMOX1 pathway signature predicts clinical benefit from immunotherapy plus tyrosine kinase inhibitor therapy in advanced renal cell carcinoma. Cancer Medicine, 12(9), 10512-10525.

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Mycobacterial Infection in Mice

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C57BL/6 or Ccr2^−/− mice were infected with ~1–5 × 10⁷ CFU M. bovis BCG intravenously. Eighteen and thirty days after infection mice were sacrificed and spleen size and weight were measured. Spleen, blood, and bone marrow were analyzed by flow cytometry for frequency of progenitors and other immune cells. Spleens were analyzed for granuloma formation by confocal microscopy. Briefly, mice were lethally anesthetized and blood was collected from the retroorbital plexus with capillaries containing anti-coagulant (EDTA), followed by a RBC lysis (eBioscience™ 1X RBC Lysis Buffer). Bone marrow was collected by flushing femoral bones with FACS buffer (1% FBS, 2 mM EDTA). Spleens were smashed through a 70 µm strainer and RBC lysis (eBioscience™ 1X RBC Lysis Buffer) was performed. After washing cells were resuspended in FACS buffer (1% FBS, 2 mM EDTA), blocked for unspecific binding with anti-FcR antibody and stained in the dark for 30 min at 4 °C. Details on antibodies used are presented in the section: Surface and intracellular staining. Cells were analyzed at a 3-laser flow cytometer (Gallios™, Beckman Coulter) and processed with the Kaluza software (v1.5, Beckman Coulter).

Lösslein A.K., Lohrmann F., Scheuermann L., Gharun K., Neuber J., Kolter J., Forde A.J., Kleimeyer C., Poh Y.Y., Mack M., Triantafyllopoulou A., Dunlap M.D., Khader S.A., Seidl M., Hölscher A., Hölscher C., Guan X.L., Dorhoi A, & Henneke P. (2021). Monocyte progenitors give rise to multinucleated giant cells. Nature Communications, 12, 2027.

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Isolation of Blood Monocyte Subsets

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Mice were anaesthetized with Ketamin/Xylazin i.p. and blood was drawn from the retro-orbital plexus. Erythrocytes were lyzed in RBC Lysis buffer solution (eBioscience™ 1X RBC Lysis buffer). The remaining cells were washed in FACS buffer and stained for CD45, CD11b, CD115, and Ly6C as described above. Ly6C^high and Ly6C^low blood monocytes were isolated with a MoFlo® Astrios™ cell sorter (Beckman Coulter).

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Liver Infiltrating Cell Isolation and Analysis

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Livers were perfused with PBS and force filtered through a 100 μm nylon cell strainer (BD Biosciences). Whole liver cell suspensions were incubated with RBC lysis buffer (eBioscience, San Diego, CA), washed with PBS-3% FBS and a small fraction was first analyzed with CD45 (BD Biosciences) or Ly6G (Biolegend, San Diego, CA) antibodies by flow cytometry to assess the specificity of infiltrated cells in the entire liver. Next, to separate infiltrated cells, whole liver cell suspensions were centrifuged at 50 g for 1 min to remove hepatocytes. Infiltrated cells were further purified by Polymorphprep^™ (CosmoBio USA, Carlsbad, CA) and layers of mononuclear and polymorphonuclear cells were recovered, following the manufacturer’s protocol. Purified infiltrated cells were incubated with RBC lysis buffer (eBioscience), washed with PBS-3% FBS, incubated with antibody cocktail, FITC-CD45 (BD Biosciences), Alexa405-CD11b (eBioscience), PEcy5.5-Ly6G (Biolegend), PE-ICAM (BD Biosciences) for 30 min on ice, and washed with PBS-3% FBS. For each time point the data obtained from Jo2-treated hBid^−/− mice (n = 4) were corrected to data from saline-treated hBid^−/− mice (n = 3). All cell suspensions were analyzed by Flow cytometer, BD LSR II (BD Biosciences), and data were analyzed by FlowJo software.

Lazic M., Eguchi A., Berk M.P., Povero D., Papouchado B., Mulya A., Johnson C.D, & Feldstein A.E. (2014). Differential regulation of inflammation and apoptosis in Fas-resistant hepatocyte-specific Bid-deficient mice. Journal of hepatology, 61(1), 107-115.

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Isolation and CFSE Labeling of Murine Splenocytes

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Spleens were collected from terminated mice, transferred to C-tubes (Miltenyi) containing 2.7 ml RPMI medium (Sigma-Aldrich) and kept on ice. Immediately before dissociation, 300 μl of a 10X Collagenase IV (1,250 U/ml; Thermo Fisher Scientific) /DNase I (10 MU; Sigma-Aldrich) enzyme cocktail was added. The C-tubes containing the spleens were placed in a gentleMACS^™ Dissociator (Miltenyi) for tissue dissociation. Subsequently, the dissociated spleens were incubated at 37°C for 10 min under constant horizontal shaking (300 rpm). After the incubation, the spleen was further disrupted using program m_spleen_03 and kept on ice until splenocyte isolation. The dissociated spleen was centrifuged for 2 min at 300 g and filtrated through a 70 μm cell strainer (Corning, NY) and centrifuged again (300 g for 5 min at 4°C). The filtrate was then incubated with 1X RBC lysis buffer (Thermo Fisher Scientific) on ice for 5 min. The suspension was neutralized by addition of 15 ml RPMI. After centrifugation (300 g for 5 min 4°C), the splenocytes were resuspended in complete RPMI medium. The cells were stained with 5 μM CFSE for 20 min at room temperature in the dark. Then, excess stain was removed, and the cells were resuspended in complete RPMI and seeded.

Wiull K., Boysen P., Kuczkowska K., Moen L.F., Carlsen H., Eijsink V.G, & Mathiesen G. (2022). Comparison of the Immunogenic Properties of Lactiplantibacillus plantarum Carrying the Mycobacterial Ag85B-ESAT-6 Antigen at Various Cellular Localizations. Frontiers in Microbiology, 13, 900922.

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Lymphocyte/Monocyte Isolation from Whole Blood

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Human whole blood samples were separated using centrifuge gradient Ficoll-paque Plus (Thermo Fisher Scientific) and the lymphocyte/monocyte layer and granulocyte/top red blood cell (RBC) layers were collected. Cells were RBC-lysed with 1XRBC lysis buffer (Thermo Fisher Scientific) and resuspended (5 × 10⁵ cells/ml) in RPMI containing 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (Pen/Strep, Thermo Fisher Scientific) for subsequent assays.

Paul A.M., Mhatre S.D., Cekanaviciute E., Schreurs A.S., Tahimic C.G., Globus R.K., Anand S., Crucian B.E, & Bhattacharya S. (2020). Neutrophil-to-Lymphocyte Ratio: A Biomarker to Monitor the Immune Status of Astronauts. Frontiers in Immunology, 11, 564950.

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