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Red blood cell lysis buffer

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Red blood cell lysis buffer is a solution used to selectively lyse or rupture red blood cells, while leaving other cell types intact. It is commonly used in cell preparation and analysis protocols to remove red blood cells from samples, allowing for the isolation and study of other cell populations.

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Lab products found in correlation

Dnase 1, by Merck Group (9 mentions) 70 μm cell strainer, by BD (7 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (6 mentions) Gentlemacs dissociator, by Miltenyi Biotec (5 mentions) Cd11b, by BioLegend (5 mentions) Lsrfortessa, by BD (5 mentions) Collagenase 1, by Merck Group (5 mentions) Cell staining buffer, by BioLegend (5 mentions) Facsdiva software, by BD (5 mentions) Hyaluronidase, by Merck Group (5 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions) Dispase 2, by Roche (4 mentions) Collagenase 2, by Worthington (4 mentions) Collagenase, by Merck Group (4 mentions) Ficoll paque plus, by GE Healthcare (4 mentions) Collagenase d, by Roche (4 mentions) Brefeldin a, by BioLegend (4 mentions) Lsrii flow cytometer, by BD (4 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (3 mentions)

232 protocols using red blood cell lysis buffer

1

Measuring ALDH Activity in Tumor Cells

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The ALDEFLUOR fluorescent assay (Stemcell Technologies) was used to measure cell-associated ALDH activity. Cells were cultured as tumorspheres, treated with the indication concentrations of cisplatin or VS-4718 for 5 days, collected by centrifugation, dissociated by trypsinization, resuspended in Aldefluor assay buffer containing ALDH substrate (BODIPY-aminoacetaldehyde), and incubated for 45 min at 37°C with or without the ALDH inhibitor diethylamino-benzaldehyde (DEAB). AldeRed substrate (EMD Millipore) was used with cells expressing GFP. Individual gates were used to determine the percentage of ALDEFLUOR-positive cells per experimental point relative to DEAB-inhibitor treated controls. For analysis of ALDH activity in ascites-associated cells, pooled isolates from peritoneal washings of each experimental group were dissociated by trypsinization, treated with red blood cell lysis buffer (Biolegend), and processed as described above.
Diaz Osterman C.J., Ozmadenci D., Kleinschmidt E.G., Taylor K.N., Barrie A.M., Jiang S., Bean L.M., Sulzmaier F.J., Jean C., Tancioni I., Anderson K., Uryu S., Cordasco E.A., Li J., Chen X.L., Fu G., Ojalill M., Rappu P., Heino J., Mark A.M., Xu G., Fisch K.M., Kolev V.N., Weaver D.T., Pachter J.A., Győrffy B., McHale M.T., Connolly D.C., Molinolo A., Stupack D.G, & Schlaepfer D.D. (2019). FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy. eLife, 8, e47327.
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2

HGSOC Organoids for PD-1 Blockade Sensitivity

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To recapitulate the tumor faithfully from their derivation and test the sensitivity to PD-1 blockade, we developed HGSOC short-term organoids culture.29 (link) The single cell suspension was incubated in 1× Red Blood Cell Lysis buffer (Biolegend) for five minutes at room temperature, and spun for three minutes at 1500 RPM. The lysis buffer was aspirated and resuspended in RPMI-1640 (10% FBS, and 1% Pen/Strep). The appropriate cell number of single cell suspension was diluted to a concentration of 6 × 106 cells/mL in RPMI-1640, 10% FBS, 1% Pen/Step, and 30 ng/mL of IL-2 (Peprotech) mixed with 15% Matrigel (Corning). 40 μL of suspension was added per well of 48 well plate. Anti-PD-1 antibody and isotype control antibody (Bioxcell) were used at a final concentration of 10 μg/mL in RPMI-1640, 10% FBS, 1% Pen/Strep, and 30 ng/mL of IL-2 for 72 hours. Protein transporter inhibitor (Invitrogen) was added to the media at a concentration of 1:500 and incubated for 2 hours prior to being harvested for flow cytometry analysis.
Cao K., Zhang G., Zhang X., Yang M., Wang Y., He M., Lu J, & Liu H. (2021). Stromal infiltrating mast cells identify immunoevasive subtype high-grade serous ovarian cancer with poor prognosis and inferior immunotherapeutic response. Oncoimmunology, 10(1), 1969075.
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3

Multiparametric Flow Cytometry Analysis

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Blood, tumor and draining lymph nodes were collected at euthanasia at different time points –tissues were collected and single cell suspensions were obtained for flow cytometry analysis. Briefly, organs were processed through a cell strainer and washed in PBS 2% FBS (Gemini) 1% PenStrep 1% NEAA (Gibco, Life Technologies). Spleen and blood samples were treated with red blood cell lysis buffer (BioLegend) before washing. Cells were stained at 1×106 cells per Antibody (0.25 µg) for 2 hours at 4°C in 100 µL FACS Buffer (1× PBS 5% FBS 0.1%). All antibodies were from BioLegend (San Diego, CA). Three panels of mouse monoclonal Abs were used; Panel 1: APC-Tigit, PerCPCy5.5-Lag3, FITC-CD8, APC-Cy7-PD1, PE-CD4; Panel 2: F480-PacBlue, FITC-Cd11b, PerCP-Cy5.5-Gr1; Panel 3: APC-CD4, PE-CD25, AF488-FoxP3. Cells were washed twice in FACS Buffer and analyzed by flow cytometry. All flow cytometry was performed at the University of Maryland Greenebaum Cancer Center Flow Cytometry Shared Services on the BD LSR II and high throughput sampler (HTS). Flow cytometry acquisition was performed using a FACS Calibur or LSRII instrument (BD Biosciences) and FACS data were analyzed using Flow Jo software (Tree Star Inc).
Chuong M., Chang E.T., Choi E.Y., Mahmood J., Lapidus R.G., Davila E, & Carrier F. (2017). Exploring the Concept of Radiation “Booster Shot” in Combination with an Anti-PD-L1 mAb to Enhance Anti-Tumor Immune Effects in Mouse Pancreas Tumors. Journal of clinical oncology and research, 5(2), 1058.
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4

Isolation of Murine Bone Marrow

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At sacrifice, femurs and tibias of NSG mice were cut with scissors, and muscles were rubbed off. BM was then flushed out with PBS using a 25G needle. After 10 min centrifugation at 10’000 rpm, BM was fully disaggregated in PBS using a 19G needle. Erythrocytes were lysed using Red Blood Cell Lysis Buffer (Biolegend) for 10 min at 37°C. BM was washed two times in PBS, and dry pellet was kept at -80°C until RNA extraction.
Mühlethaler-Mottet A., Liberman J., Ascenção K., Flahaut M., Balmas Bourloud K., Yan P., Jauquier N., Gross N, & Joseph J.M. (2015). The CXCR4/CXCR7/CXCL12 Axis Is Involved in a Secondary but Complex Control of Neuroblastoma Metastatic Cell Homing. PLoS ONE, 10(5), e0125616.
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5

Immune response assessment following influenza exposure

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Two nasal swabs (one per nostril) were taken daily following infection with wt H1N1 and immunization with LAIV and following subsequent challenge with wt H1N1. The swabs were placed into 2 ml of virus transport medium comprising tissue culture medium 199 (Sigma-Aldrich, UK) supplemented with 25 mM Hepes, 0.035% sodium bicarbonate, 0.5% BSA, penicillin, streptomycin and nystatin, vortexed, centrifuged to remove debris and stored at −80°C for subsequent virus titration. Serum and heparin anti-coagulated blood samples were collected at the start of the study (prior to LAIV immunization or wt H1N1 pre-exposure), before challenge and four dpc at post-mortem. Heparin blood samples were diluted 1:1 in PBS before density gradient centrifugation at 1,200 × g for 30 min over Histopaque® 1.083 g/ml (Sigma-Aldrich, UK). PBMC were harvested from the interface, washed and red blood cells lysed with Red Blood Cell Lysis Buffer (BioLegend, UK), washed again and cryopreserved in FCS (Gibco) with 10% DMSO (Sigma-Aldrich, UK). Broncho-alveolar lavage (BAL) and tracheobronchial lymph nodes (TBLN) were processed as previously described (38 (link)).
Holzer B., Morgan S.B., Martini V., Sharma R., Clark B., Chiu C., Salguero F.J, & Tchilian E. (2019). Immunogenicity and Protective Efficacy of Seasonal Human Live Attenuated Cold-Adapted Influenza Virus Vaccine in Pigs. Frontiers in Immunology, 10, 2625.
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6

Multicolor Flow Cytometry Analysis

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Bone marrow and peripheral blood cells were treated with red blood cell lysis buffer (Biolegend) for 15 min in the dark and were then stained for anti-CD90 (HIS51) fluorescein isothiocyanate (FITC), anti-CD11b (WT.5) allophycocyanin (APC) (BD Biosciences), anti-CD117 (2B8) APC, anti-CD19 (1D3) PE-cyanine 7 (PE/Cy7) (eBiosciences), and anti-CD3 (17A2) APC-cyanine 7 (APC/Cy7) (BioLegend). Flow cytometric analyses were carried out using an LSR Fortessa flow cytometer equipped with FACS Diva 6.2 Software (BD Biosciences). Data were analyzed with a FlowJo 9.8.2 software.
Alvarez-Martins I., Remédio L., Matias I., Diogo L.N., Monteiro E.C, & Dias S. (2016). The impact of chronic intermittent hypoxia on hematopoiesis and the bone marrow microenvironment. Pflugers Archiv, 468, 919-932.
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7

Isolation and Preparation of Splenic Cells

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After euthanasia, spleens were removed from mice and transferred to a 70-μm cell strainer placed on top of a 50 mL tube. The tissue was pushed through the strainer using a 10 mL syringe plunger and washed with 10 mL ice-cold DMEM (Corning®) to make a single cell suspension. Cells were collected by centrifuge (400 g, 5 min), and red blood cells were removed by Red Blood Cell Lysis Buffer (Biolegend). Splenic cells were then resuspended in DMEM, counted on TC20™ Automated Cell Counter (Bio-Rad Laboratories) and kept on ice for flow cytometry staining.
Wang J., Leavenworth J., Hjelmeland A.B., Smith R., Patel N., Borg B., Si Y, & King P.H. (2019). Deletion of the RNA regulator HuR in tumor associated microglia and macrophages stimulates antitumor immunity and attenuates glioma growth. Glia, 67(12), 2424-2439.
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8

Isolation of Primary Mouse Cell Types

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Primary Mouse Embryonic Fibroblasts (MEFs) were generated from E13.5 embryos. After removing the placenta, yolk sac, head and the dark red organs, embryos were finely minced and digested for 20 min in 0.25% trypsin. Single cell suspension was then obtained by pipetting up and down the digested embryos. Mouse Dermal Fibroblasts (MDFs) were isolated as described in (Etemadi et al., 2015 (link)). To generate Bone Marrow Derived Macrophages (BMDMs), bone marrow cells from tibia and femur of 2 month old mice were seeded in non-coated Petri dishes and cultured for 6 days in Dulbecco’s modified Eagle medium + 10% fetal bovine serum + 20% (v/v) L929 mouse fibroblast conditioned medium. Keratinocytes were isolated as described in (Lichti et al., 2008 (link)). Splenocytes were isolated from 2 month old mice. Mouse spleens were mashed through a cell strainer into the Petri dish using the plunger end of a syringe. Cells were then washed once in cold PBS and treated with 1X Red Blood Cell Lysis Buffer (BioLegend, Cat N 420301) for 5 min on ice. Cells were then washed again in PBS and counted.
Liccardi G., Ramos Garcia L., Tenev T., Annibaldi A., Legrand A.J., Robertson D., Feltham R., Anderton H., Darding M., Peltzer N., Dannappel M., Schünke H., Fava L.L., Haschka M.D., Glatter T., Nesvizhskii A., Schmidt A., Harris P.A., Bertin J., Gough P.J., Villunger A., Silke J., Pasparakis M., Bianchi K, & Meier P. (2019). RIPK1 and Caspase-8 Ensure Chromosome Stability Independently of Their Role in Cell Death and Inflammation. Molecular Cell, 73(3), 413-428.e7.
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9

Comprehensive Flow Cytometry Analysis

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All samples were analyzed using a NovoCyteTM (ACEA Biosciences), LSR Fortessa, or C6 flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR, USA). The antibodies used included anti-MSLN-biotin (clone MB), Streptavidin-APC, anti-human CCR7-APC (clone 3D12), anti-human CD62L-PE (clone DREG-56), anti-human CD45RA-APC (clone HI100), anti-human CD45RO-PE (clone UCHL1), anti-human TIM3-PE (clone F38-2E2), anti-human LAG3-PerCP/Cy5.5 (clone 11C3C65), anti-human PD-1-APC (clone NAT105), anti-human CD27-PE (clone M-T271), anti-human CD28-APC (clone CD28.2), anti-human CD25-PE (clone BC96), anti-human CD69-APC (clone FN50), anti-human CD107a-APC (clone H4A3), anti-human CD3-APC (clone UCHT1), anti-human CD4-PerCP/Cy5.5 (clone OKT4), anti-human CD8-PE (clone OKT8), mouse IgG2a isotype control-APC (clone RMG2a-62), mouse IgG1kappa isotype control-PE, mouse IgG1kappa isotype control-PErCP/Cy5.5, and mouse IgG1kappa isotype control-APC (clone MOPC-21) (Biolegend, San Diego, CA, USA). All FACS-related staining procedures were performed on ice for 30 min, and cells were then washed with PBS containing 1% FBS before cytometry analysis. PB, spleen (SP), and tumor samples from mouse xenografts were treated with red blood cell lysis buffer (Biolegend), and the cells were stained with the corresponding antibodies.
Lv J., Zhao R., Wu D., Zheng D., Wu Z., Shi J., Wei X., Wu Q., Long Y., Lin S., Wang S., Wang Z., Li Y., Chen Y., He Q., Chen S., Yao H., Liu Z., Tang Z., Yao Y., Pei D., Liu P., Zhang X., Zhang Z., Cui S., Chen R, & Li P. (2019). Mesothelin is a target of chimeric antigen receptor T cells for treating gastric cancer. Journal of Hematology & Oncology, 12, 18.
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10

Tissue Harvest and Dissociation

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Spleen, inguinal LN and thymus were harvested and dissociated mechanically. Lung tissue was digested with Collagenase I (Life Technologies) for 30 min at 37°C. Single cell suspensions were treated with red blood cell lysis buffer (BioLegend).
Gupta P.K., Wagner S.R., Wu Q, & Shilling R.A. (2016). Th17 cells are not required for maintenance of IL-17A producing γδ T cells in vivo. Immunology and cell biology, 95(3), 280-286.
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