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Red blood cell lysis

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Red blood cell lysis buffer is a solution used to lyse or break down red blood cells, separating them from other blood components such as white blood cells and platelets. This allows for the isolation and analysis of specific cell types within a blood sample.

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Lab products found in correlation

Countess 2 automated cell counter, by Thermo Fisher Scientific (2 mentions) Countess automated cell counter, by Thermo Fisher Scientific (2 mentions) Hi fbs, by Sartorius (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Moflo astrios eq, by Beckman Coulter (1 mentions) Dnase 1, by Merck Group (1 mentions) Liberase tl, by Roche (1 mentions) Lps sc 3535, by Santa Cruz Biotechnology (1 mentions) Il 4 214 14, by Thermo Fisher Scientific (1 mentions) Cell strainer, by Corning (1 mentions) Intracellular staining permeabilization solution, by BioLegend (1 mentions) Lsrfortessa flow cytometer, by BD (1 mentions) Heparin, by Merck Group (1 mentions) Dulbecco modified eagle medium (dmem), by Roche (1 mentions) Rbc lysis buffer, by BioLegend (1 mentions) Collagenase a, by Merck Group (1 mentions) Percoll, by GE Healthcare (1 mentions) Facs lysing solution, by BD (1 mentions) Dnase 1, by Roche (1 mentions) Collagenase d, by Roche (1 mentions)

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Red blood cells lysis buffer

11 protocols using red blood cell lysis

1

Isolation and Characterization of Lung and Peritoneal Cells

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Lungs were minced with scissors and digested for 30 minutes at 4 °C in RPMI1640 with 2.5 mg/ml collagenase I and 0.25 mg/ml DNase I. The digest was sequentially passed and mashed through a 70 μm cell strainer and the strainer washed with FACS buffer. The cell suspension was filtered with a 50 μm cell strainer and red blood cells were lysed. Cells were counted using Countess automated cell counter (Invitrogen) and further process for multicolour flow cytometry. Cells were kept on ice until use or analysis.
PBS or IL-4c injected mice were killed by cervical dislocation, peritoneal exudate cells (PECs) were harvested by lavage of the peritoneal cavity with sterile PBS. Total peritoneal exudate cells were counted after red blood cell lysis (Biolegend) using Countess II automated cell counter (Invitrogen) and further process for magnetic-activated cell sorting (MACS). Cells were kept on ice until use or analysis.
Bidault G., Virtue S., Petkevicius K., Jolin H.E., Dugourd A., Guénantin A.C., Leggat J., Mahler-Araujo B., Lam B.Y., Ma M.K., Dale M., Carobbio S., Kaser A., Fallon P.G., Saez-Rodriguez J., McKenzie A.N, & Vidal-Puig A. (2021). SREBP1-induced fatty acid synthesis depletes macrophages antioxidant defences to promote their alternative activation. Nature metabolism, 3(9), 1150-1162.
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2

Isolation and Analysis of Single Cells

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The obtained tissues were cut into small pieces and shaken with Liberase TL (100 U/mL; Roche) with DNase I (100 U/mL; Sigma) in a 37 °C water bath for 20 min. Subsequently, the cell suspensions were passed through a 70-μm cell strainer to produce single cells. The collected peripheral blood samples were subjected to red blood cell lysis (BioLegend) and then washed twice with PBS. Single-cell suspensions, tissues, and blood samples were incubated with the appropriate antibodies (Additional file 1: Table S1) for 15 min in the dark. The cells were washed twice, resuspended in PBS, and analyzed by MoFlo Astrios EQ (Beckman Coulter) for at least three independent experiments. The data were analyzed using FlowJo V10.0 (Tree Star). Information on the antibodies is listed in Additional file 1: Table S1.
Liu H., Zhu X., Cao X., Chi A., Dai J., Wang Z., Deng C, & Zhang M. (2021). IL-1β-primed mesenchymal stromal cells exert enhanced therapeutic effects to alleviate Chronic Prostatitis/Chronic Pelvic Pain Syndrome through systemic immunity. Stem Cell Research & Therapy, 12, 514.
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3

Isolation and Activation of Mouse Splenocytes

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For mouse cells, whole spleens were extracted from donor (CD45.1) mice and were mechanically crushed in PBS to be filtered in a 70 μm Cell Strainer (Corning). Following Red Blood Cell lysis (420301, Biolegend), cells were plated at 3.0E6 cells/ml in 1640 RPMI (01-100-1A, Biological Industries) supplemented with 10% HI FBS (04-127-1A, Biological Industries), 50 μM β-Mercaptoethanol, P/S (03-031-1B, Biological Industries), 10 μg/ml LPS (sc-3535, SantaCruz Biotechnology) and 10 ng/ml IL4 (214-14, Peprotech) was added immediately after extraction.
Cells were cultured 16–24 h and washed in PBS before transfections and plated in the same activation medium without P/S for 8–16 h following electroporations. Parameters were 1350 v 30 ms 1 pulse at 4.0E5 cells/μl in buffer R for 10 μl tips. For RNP: Cas9 (1081059, IDT) and gRNA (IDT) complexes assemblies were generated 20 min prior of transfection with 18.3 pmol Cas9 and 38 pmol gRNA per 1E06 cells. Transductions were performed no later than 5 min following electroporation with a 50,000 MOI of rAAV-DJ and cells were analyzed 2 days following electroporation. For co-culture with CD40LB feeders, splenic lymphocytes were seeded as previously described17 (link).
Nahmad A.D., Raviv Y., Horovitz-Fried M., Sofer I., Akriv T., Nataf D., Dotan I., Carmi Y., Burstein D., Wine Y., Benhar I, & Barzel A. (2020). Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion. Nature Communications, 11, 5851.
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4

Prime-Boost Immunization and HIV Drug Efficacy

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LVs and VLPs were injected subcutaneously into the right flank of mice. Prime-boost immunizations contained between 25 and 50 ng of p24. All vectors were normalized to equivalent amounts of OVA and/or p24 by ELISA. Unimmunized mice received equal volume of injections of phosphate-buffered saline (PBS). Blood samples were lysed with red blood cell lysis (BioLegend) before analysis. For the tumor experiments, mice received 5 × 106 EL4 or E.G7 cells injected subcutaneously into the opposing flanks of the mice. Tumor size was measured and shown as a product of the two largest perpendicular diameters a × b (in square millimeters). Tablets containing efavirenz (600 mg), emtricitabine (200 mg), and tenofovir disoproxil fumarate (300 mg; Cipla) were crushed, resuspended in PBS containing 1% (v/v) dimethyl sulfoxide, filtered through a 0.22-μm filter, and stored in aliquots at −80°C. RTIs were added to the fresh drinking water of mice containing efavirenz (10 mg ml−1), emtricitabine (3.6 mg ml−1), and tenofovir (5.4 mg ml−1). Fresh water containing RTIs was replaced two times a week. Mice receiving no RTIs were given similar volumes of drinking water and replaced accordingly. Mice were initiated on RTIs 1 week before immunization and continued throughout the duration of the experiment.
Kim J.T., Liu Y., Kulkarni R.P., Lee K.K., Dai B., Lovely G., Ouyang Y., Wang P., Yang L, & Baltimore D. (2017). Dendritic cell–targeted lentiviral vector immunization uses pseudotransduction and DNA-mediated STING and cGAS activation. Science immunology, 2(13), eaal1329.
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5

Profiling Immune Landscape in the Tumor Microenvironment

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The detection of apoptosis and the cell cycle was performed as described in previous literature [15 (link)]. For the detection of TME, the peripheral blood (PB), tumor, and spleen of mice were made into single-cell suspensions. After red blood cell lysis (Biolegend), cells were counted and, at a number of 3–5 × 106 per tube were incubated with fluorescently labeled antibody cocktail (30 min, 4℃, in the dark). For cell surface staining, the single-cell suspensions were incubated with the antibodies on ice for 30 min. For intracellular staining, cells were fixed and then permeabilized with intracellular staining/permeabilization solution (Bio Legend, San Diego, CA, USA) and then stained with intracellular antibodies on ice for 30 min. Data were collected using an LSRFortessa flow cytometer (BD Bioscience) and analyzed with FlowJo 10 software. Details of the antibodies are shown in Additional file 5: Table S2.
Qiu J., Xia Y., Bao Y., Cheng J., Liu L, & Qian D. (2024). Silencing PinX1 enhances radiosensitivity and antitumor-immunity of radiotherapy in non-small cell lung cancer. Journal of Translational Medicine, 22, 228.
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6

Isolation of Immune Cells from Various Tissues

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Peripheral blood samples (10-30 μl) were obtained by facial vein puncture in 1 μl heparin (10U/μl, Sigma). Blood erythrocytes were lysed and cells fixed using BD FACS Lysing Solution. For other tissues that required red blood cell lysis, RBC Lysis buffer (Biolegend) was used. For the isolation of immune cells from the CNS, mice were anesthetized and transcardially perfused with PBS. Brain and spinal cord were minced, digested in DMEM (ThermoFisher) with Collagenase A (1mg/mL, Sigma) and DNaseI (0.1 mg/mL, Roche) for 1 h at 37°C and homogenized using 70-μm cell strainers (BD). Leukocytes were separated using a discontinuous Percoll (GE healthcare) gradient (30% / 70%, 2500 RPM, 30 min, 4°C). Bone marrow cells were flushed from 1 femur per mouse. For analysis of DC and monocyte populations, axillary and brachial lymph nodes or spleen were cut into small pieces and digested in DMEM with collagenase D (1mg/mL, Roche) and DNaseI (10μg/mL, Roche) for 30-35 min at 37°C under constant pipetting. For studies of all other immune cells, lymph nodes or spleens were mashed through a 70 μm cell strainer. For splenocytes, red blood cell lysis was performed (BioLegend).
Spiljar M., Steinbach K., Rigo D., Suárez-Zamorano N., Wagner I., Hadadi N., Vincenti I., Page N., Klimek B., Rochat M.A., Kreutzfeldt M., Chevalier C., Stojanović O., Bejuy O., Colin D., Mack M., Cansever D., Greter M., Merkler D, & Trajkovski M. (2021). Cold exposure protects from neuroinflammation through immunologic reprogramming. Cell Metabolism, 33(11), 2231-2246.e8.
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7

Efficient B Cell Genome Editing

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For human cells, whole blood was obtained with donor consent from the Israeli blood bank (Magen David Adom, Sheiba Medical Center) in accordance with Tel Aviv University Review Board, and PBMCs were extracted using Lymphocyte Separation Medium (0850494-CF, mpbio). Remnants of red blood cells were lysed using Red Blood Cell lysis (420301, Biolegend). B cells were enriched using the negative selection Easysep human B cell isolation kit (17954, Stemcell) and plated in 1640 RPMI (01-100-1A, Biological Industries) supplemented with 10% HI FBS (04-127-1 A, Biological Industries), 50 μM β-Mercaptoethanol, P/S (03-031-1B, Biological Industries), 2 μg/ml RP105 (312907, Biolegend) and 10 ng/ml IL7 (200-07, Peprotech).
For human primary cells, electroporation parameters were: 1750 v 20 ms 1 pulse in a Neon electroporation system (Invitrogen) at 4.0E5 cells/μl in buffer R using 10 μl tips. For RNP, Cas9 (1081059, IDT) and gRNA (IDT): complexes assemblies were generated 20 min prior to transfection with 18.3 pmol Cas9 and 66 pmol gRNA per 1E06 cells. Transductions were performed no later than 5 min following electroporation with a 10,000 MOI of rAAV-6. Efficiency of editing was determined 2 days following electroporation.
Nahmad A.D., Raviv Y., Horovitz-Fried M., Sofer I., Akriv T., Nataf D., Dotan I., Carmi Y., Burstein D., Wine Y., Benhar I, & Barzel A. (2020). Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion. Nature Communications, 11, 5851.
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8

Isolation and Characterization of Lung and Peritoneal Cells

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Lungs were minced with scissors and digested for 30 minutes at 4 °C in RPMI1640 with 2.5 mg/ml collagenase I and 0.25 mg/ml DNase I. The digest was sequentially passed and mashed through a 70 μm cell strainer and the strainer washed with FACS buffer. The cell suspension was filtered with a 50 μm cell strainer and red blood cells were lysed. Cells were counted using Countess automated cell counter (Invitrogen) and further process for multicolour flow cytometry. Cells were kept on ice until use or analysis.
PBS or IL-4c injected mice were killed by cervical dislocation, peritoneal exudate cells (PECs) were harvested by lavage of the peritoneal cavity with sterile PBS. Total peritoneal exudate cells were counted after red blood cell lysis (Biolegend) using Countess II automated cell counter (Invitrogen) and further process for magnetic-activated cell sorting (MACS). Cells were kept on ice until use or analysis.
Bidault G., Virtue S., Petkevicius K., Jolin H.E., Dugourd A., Guénantin A.C., Leggat J., Mahler-Araujo B., Lam B.Y., Ma M.K., Dale M., Carobbio S., Kaser A., Fallon P.G., Saez-Rodriguez J., McKenzie A.N, & Vidal-Puig A. (2021). SREBP1-induced fatty acid synthesis depletes macrophages antioxidant defences to promote their alternative activation. Nature metabolism, 3(9), 1150-1162.
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9

Isolation and Sorting of Mouse Adipose Cells

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For scRNA-seq, flow cytometry analysis, and flow cytometry sorting, mouse Epi/VAT biopsies were minced and digested in 4–6 mg/mL collagenase I (Sigma-Aldrich), 4–6 mg/mL collagenase II (Roche), and 1% BSA (Sigma-Aldrich) in Hanks Balanced Salt Solution (Sigma-Aldrich) at 37°C for 45 minutes with agitation (180 rpm), then, strained through a 70-μm strainer in PBS with 0.1% BSA. The cell suspension was centrifuged at 300 ×g for 5 minutes, the supernatant removed, and the cell pellet resus-pended in PBS with 0.1% BSA. Red blood cell lysis was performed (BioLegend), followed by wash and centrifugation (300 ×g, 5 minutes), resuspension in PBS, and straining through a 40 μm filter. For scRNA-seq, the SVF suspension was blocked in the anti-mouse CD16/32 antibody (BioLegend) for 30 minutes at 4°C followed by antibody incubation with CD11b-Alexa Fluor 488 and CD45-APC (BioLegend) for 30 at 4°C. Cells were then washed twice with PBS and sorted using fluorescence-activated cell sorting (FACS) (SY3200 Synergy, Sony Biotechnology).
Harasymowicz N.S., Rashidi N., Savadipour A., Wu C.L., Tang R., Bramley J., Buchser W, & Guilak F. (2021). Single-cell RNA sequencing reveals the induction of novel myeloid and myeloid-associated cell populations in visceral fat with long-term obesity. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 35(3), e21417.
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10

Spleen Cell Isolation Protocol

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Harvested spleens were mechanically dissociated by using 1 ml syringe plungers to mash the spleens through 30-µm strainers. Red blood cell lysis (BioLegend #420302) was performed on a single cell suspension for 1 min before further downstream processing.
Adu-Berchie K., Brockman J.M., Liu Y., To T.W., Zhang D.K., Najibi A.J., Binenbaum Y., Stafford A., Dimitrakakis N., Sobral M.C., Dellacherie M.O, & Mooney D.J. (2023). Adoptive T cell transfer and host antigen-presenting cell recruitment with cryogel scaffolds promotes long-term protection against solid tumors. Nature Communications, 14, 3546.
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