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Rbc lysis

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RBC lysis is a laboratory equipment used to disrupt red blood cells (RBCs) in a sample. It disrupts the cell membrane of RBCs, allowing the release of cellular contents for further analysis or processing. The core function of RBC lysis is to prepare samples by removing RBCs, which can interfere with the analysis of other cell types or analytes of interest.

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Lab products found in correlation

Anti il 10, by BioLegend (2 mentions) Anti ly6c, by Bio-Rad (2 mentions) Ionomycin phorbol 12 myristate 13 acetate brefeldin a, by BioLegend (2 mentions) Foxp3 fix perm, by BioLegend (2 mentions) Anti il 17, by BioLegend (2 mentions) Anti cd4, by Thermo Fisher Scientific (2 mentions) Anti cd4, by BioLegend (2 mentions) Anti cd11b, by BioLegend (2 mentions) Anti ly6g, by BioLegend (2 mentions) Anti cd8, by BioLegend (2 mentions) Anti cd44, by BioLegend (2 mentions) Anti cd62l, by BioLegend (2 mentions) Facsaria 3, by BD (1 mentions) Easysep mouse pe positive selection kit 2, by STEMCELL (1 mentions) Easysep mouse t cell isolation kit, by STEMCELL (1 mentions) X rad 320, by Precision X-Ray (1 mentions) Ls004188, by Worthington (1 mentions) Ls002592, by Worthington (1 mentions) 70 μm cell strainer, by BD (1 mentions) Soybean trypsin inhibitor, by Merck Group (1 mentions)

12 protocols using rbc lysis

1

Isolating and Analyzing Mouse Spleen, Thymus, and Bone Marrow Cells

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The spleen and thymus were isolated. Each organ was placed inside a 100 μm cell strainer on top a 50 mL conical tube. Using the plunger end of a 5 mL syringe the tissues were pulverized. The cells were collected in the 50 mL tube by rinsing the plunger and strainer with 10 mL of HBSS containing 3% fetal calf serum. The cells were pelleted by centrifugation and any contaminating red blood cells were lysed and removed by additional centrifugation. Between 4 × 105 and 1 × 106 viable cells were used for staining with each antibody cocktail.
The femora and tibiae from both sides of the mouse were isolated, opened at both ends and flushed with PBS using a 27G needle attached to 1 cc syringe. The eluent was collected in a 50 mL conical tube. The cells were pelleted by centrifugation at 500 ×g for 10 min at 4 °C and any contaminating red blood cells were lysed using 1X RBC Lysis (eBioscience, Catalog #00433357) according to the manufacturer’s protocol and removed by additional centrifugation. Between 105 and 1 × 106 viable bone marrow cells were used for staining with each antibody cocktail (Supplementary table 1).
All antibodies used in flow cytometry analysis were purchased from BD Bioscience pre-conjugated with the fluorophore.
Wilson E.M., Bial J., Tarlow B., Bial G., Jensen B., Greiner D.L., Brehm M.A, & Grompe M. (2014). Extensive double humanization of both liver and hematopoiesis in FRGN mice. Stem cell research, 13(3 Pt A), 404-412.
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2

Isolating and Analyzing Mouse Spleen, Thymus, and Bone Marrow Cells

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The spleen and thymus were isolated. Each organ was placed inside a 100 μm cell strainer on top a 50 mL conical tube. Using the plunger end of a 5 mL syringe the tissues were pulverized. The cells were collected in the 50 mL tube by rinsing the plunger and strainer with 10 mL of HBSS containing 3% fetal calf serum. The cells were pelleted by centrifugation and any contaminating red blood cells were lysed and removed by additional centrifugation. Between 4 × 105 and 1 × 106 viable cells were used for staining with each antibody cocktail.
The femora and tibiae from both sides of the mouse were isolated, opened at both ends and flushed with PBS using a 27G needle attached to 1 cc syringe. The eluent was collected in a 50 mL conical tube. The cells were pelleted by centrifugation at 500 ×g for 10 min at 4 °C and any contaminating red blood cells were lysed using 1X RBC Lysis (eBioscience, Catalog #00433357) according to the manufacturer’s protocol and removed by additional centrifugation. Between 105 and 1 × 106 viable bone marrow cells were used for staining with each antibody cocktail (Supplementary table 1).
All antibodies used in flow cytometry analysis were purchased from BD Bioscience pre-conjugated with the fluorophore.
Wilson E.M., Bial J., Tarlow B., Bial G., Jensen B., Greiner D.L., Brehm M.A, & Grompe M. (2014). Extensive double humanization of both liver and hematopoiesis in FRGN mice. Stem cell research, 13(3 Pt A), 404-412.
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3

Bone Marrow Transplantation in Mice

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Six- to seven-week-old WT or Il1a−/− recipient mice were irradiated at a lethal, single dose of 850 cGy (X-rad 320). Donor bone marrow cells were isolated from hind leg femurs and tibias of WT or Il1a−/− mice. Cells were treated with 1× RBC lysis (eBioscience) for 2 min and then washed with sterile PBS. Cells were counted and resuspended at 1 × 107 cells/ml PBS. Irradiated recipient mice were anesthetized with isoflurane and transplanted with 100 μl (1 × 106 cells) of bone marrow donor cells by retro-orbital injection. Transplanted mice were kept on an antibiotic chow diet (UNI-PRIM) for 3 wk before returning to regular chow for 1–2 wk before infection.
Ratitong B., Marshall M.E., Dragan M.A., Anunciado C.M., Abbondante S, & Pearlman E. (2022). Differential Roles for IL-1α and IL-1β in Pseudomonas aeruginosa Corneal Infection. Journal of immunology (Baltimore, Md. : 1950), 209(3), 548-558.
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4

Competitive Repopulation Assay for Hematopoietic Stem Cells

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Competitive repopulation assays were performed as described previously [26 (link), 27 (link)]. Specifically, for first transplantation, the cells (1 × 106 BMCs) were harvested from the donor Ly5.1 mice and then mixed with 2 × 105 competitive BMCs from Ly5.2 mice. The mixed cells were transplanted into lethally irradiated (9.5 Gy TBI) Ly5.2 recipient mice though lateral canthus vein injection. For second transplantation, CD45.2 cells (1 × 106 BMCs) sorted from 1st transplantation recipient mice accompany with 2 × 105 competitive BMCs from Ly5.2 mice were transplanted into lethally irradiated (9.5 Gy) Ly5.2 s recipient mice. To analyze the donor cells engraftment, peripheral blood was harvested at 8 and 16 weeks after first transplantation and 16 weeks after second transplantation. The red blood cells were lysed by 1 × RBC lysis (eBioscience Inc. San Diego, CA, USA), and the blood samples were stained with anti-CD45.1-FITC, anti-CD45.2-PE, anti-CD3-APC, anti-B220-PerCP, and anti-Gr-1 and CD11b-PE/Cy7 and were analyzed by BD FACS AriaIII.
Li C., Lu L., Zhang J., Huang S., Xing Y., Zhao M., Zhou D., Li D, & Meng A. (2015). Granulocyte colony-stimulating factor exacerbates hematopoietic stem cell injury after irradiation. Cell & Bioscience, 5, 65.
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5

Endogenous BM Ablation and Reconstitution

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To ablate endogenous BM, WT B6 mice were irradiated to 9 Gy using an X-RAD 320 (Precision X-Ray). After a 6-hour rest period, mice were transplanted with 4 × 106 donor BM cells via i.v. tail vein injection. BM cells were flushed from the femurs of donor WT B6 or IL-12Rb2–KO mice. Marrow was subjected to RBC lysis (Thermo Fisher Scientific) and filtered (30 μM). For BM mixing studies, untouched WT BM CD3+ cells were isolated from WT B6 donor marrow with the EasySep Mouse T cell Isolation Kit (Stemcell) from Stemcell used per manufacturer recommendations. Untouched IL-12Rb2–KO CD3– cells were isolated from IL-12Rb2–KO donor marrow by using positive selection to remove CD3+ cells with a PE-conjugated CD3 primary antibody (BioLegend), followed by positive selection of PE-labeled cells with the EasySep Mouse PE Positive Selection Kit II (Stemcell Technologies) used per manufacturer recommendations. Transplanted mixed BM consisted of 10% CD3+ and 90% CD3– cells. Mice were allowed 3 weeks to engraft new marrow before being transplanted with MOC22 cells and used to in vivo studies.
Hong Y., Robbins Y., Yang X., Mydlarz W.K., Sowers A., Mitchell J.B., Gulley J.L., Schlom J., Gameiro S.R., Sievers C, & Allen C.T. (2022). Cure of syngeneic carcinomas with targeted IL-12 through obligate reprogramming of lymphoid and myeloid immunity. JCI Insight, 7(5), e157448.
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6

Isolation of Tumor-Infiltrating Lymphocytes

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Single-cell suspensions were prepared from isolated tumors and spleens. Spleens were mechanically disrupted, re-suspended in 1% FBS/PBS and processed following RBC lysis (#00-4300-54, Thermo Fisher). For isolation of tumor-infiltrating lymphocytes, tumor tissue was minced into 1 to 2 mm pieces and digested with collagenase IV (1.25mg/mL; #LS004188, Worthington), 0.1% soybean trypsin inhibitor (#T9128, Sigma), hyaluronidase (1mg/mL; #LS002592, Worthington) and DNase I (100 mg/mL; #LS002007, Worthington) in DMEM for 30 min at 37°C. Cell suspensions were passed through a 70μm cell strainer (Falcon) and resuspended in 2% FBS DMEM media. Lymphocytes were isolated from processed tumor tissues by Ficoll density gradient centrifugation. For some experiments, isolation of total CD45+ leukocytes by magnetic-activated cell sorting (#130-052-301, MACS Miltenyi Biotec) was performed on the leukocyte-enriched fraction according to manufacturer’s protocol, with purity of >90% achieved. Cells were collected in complete RPMI media containing 10% FCS with 1% PS.
Li S., Mirlekar B., Johnson B.M., Brickey W.J., Wrobel J.A., Yang N., Song D., Entwistle S., Tan X., Deng M., Cui Y., Li W., Vincent B.G., Gale M J.r., Pylayeva-Gupta Y, & Ting J.P. (2022). STING-induced regulatory B cells compromise NK function in cancer immunity. Nature, 610(7931), 373-380.
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7

IL-6 Signaling Pathway Modulation

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Mononuclear cells were obtained from wild-type bone marrow cells after RBC lysis (eBioscience). Cells were resuspended in serum-free RPMI 1640 medium; 1 × 106 cells were then seeded in each well of a 12-well plate. Cells were treated with anti–mouse IL-6 antibody (BE0046, InVivoMAb) or mouse gp130 Fc chimera for 1 hour under culture condition. Mouse IgG1 isotype control and control Fc fusion protein (Enzo Life Sciences) were used as negative controls for anti–mouse IL-6 antibody and mouse gp130 Fc chimera, respectively. Cells were challenged with mouse recombinant IL-6 at a final concentration of 10 ng/mL for 15 minutes and then harvested with RIPA buffer for Western blot analyses.
Mei Y., Ren K., Liu Y., Ma A., Xia Z., Han X., Li E., Tariq H., Bao H., Xie X., Zou C., Zhang D., Li Z., Dong L., Verma A., Lu X., Abaza Y., Altman J.K., Sukhanova M., Yang J, & Ji P. (2022). Bone marrow–confined IL-6 signaling mediates the progression of myelodysplastic syndromes to acute myeloid leukemia. The Journal of Clinical Investigation, 132(17), e152673.
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8

Murine Lymphoid Tissue Isolation

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After euthanization, murine spleen, mesenteric lymph nodes and inguinal lymph nodes were harvested and crushed gently on a 70 µm cell strainer using a 1 mL syringe plunger. After two washes in fluorescence-activated cell sorter (FACS) buffer (PBS containing 1% FBS and 2 mM EDTA), the cells were subjected to red blood cell (RBC) lysis (eBioSciences)) for 5–7 min, followed by two washes with FACS buffer. The single-cell preparations were resuspended in FACS buffer containing 10 µg/mL Fc receptor block and incubated for 15 min. For dendritic cell (DC), macrophages and B cell analysis, lymph node and splenocytes were stained with antibodies against the following multiple surface antigens: anti-CD11c, anti-MHC-II, F4/80, CD11b and CD19. For T cells, natural killer (NK) and natural killer T cell (NKT) cell analysis, cells were stained with anti CD3, anti CD4, anti CD8, anti NK1.1 and anti NKp46 antibodies. Dead cells were stained with Live/Dead fixable near-infrared staining kit (Life Technologies) in PBS for 30 min at 4°C. ice. Surface-stained samples were fixed with FACS buffer containing 0.1% formaldehyde before acquisition. Data analysis was performed using FlowJo software (Treestar).
Shenderov E., Kandasamy M., Gileadi U., Chen J., Shepherd D., Gibbs J., Prota G., Silk J.D., Yewdell J.W, & Cerundolo V. (2021). Generation and characterization of HLA-A2 transgenic mice expressing the human TCR 1G4 specific for the HLA-A2 restricted NY-ESO-1157-165 tumor-specific peptide. Journal for Immunotherapy of Cancer, 9(6), e002544.
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9

Multiparametric Flow Cytometry of Immune Cells

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Spleens were sieved (70 µm), before washing [phosphate-buffered saline (PBS); 350 g, 5
min], re-sieving (40 µm), RBC lysis (eBioscience), washing, and resuspension in FACS
buffer (1% BSA, 0.05% NaN3, in PBS) or full RPMI 1640. Cells in FACS buffer
were Fc blocked (1:100; BioLegend; 10 min, RT) before staining for T-cell activation with:
anti-CD4 (1:800; eBioscience), anti-CD8 (1:100), anti-CD62L (1:80), anti-CD44 (1:400; all
BioLegend; 20 min, RT); or for Tregs with: anti-CD4 (1:800), anti-CD25 (1:80; BioLegend;
20 min, RT), before washing, fixation, permeabilization (FOXP3 Fix/Perm; BioLegend), then
anti-FOXP3 (1:20; BioLegend; 30 min, RT). Splenocytes in RPMI were treated
±ionomycin/phorbol 12-myristate 13-acetate/brefeldin A (1:500; BioLegend), incubated (5 h,
37°C), washed, resuspended in FACS buffer, Fc blocked, stained with anti-CD4 or anti-CD8
(20 min, RT), washed, fixed, permeabilized (BioLegend), then anti-IL-10, anti-IL-17, and
anti-interferon γ (IFNγ; all 1:100; BioLegend; 30 min, RT). For neutrophil, monocyte or
Ly6C+ cells, whole blood (ethylenediaminetetraacetic acid) was stained with
anti-CD115 (1:100; eBioscience), anti-CD11b (1:800), anti-Ly6G (1:80; both BioLegend),
anti-Ly6C (1:400; AbD Serotec, Hercules, CA), at RT for 30 min, before RBC lysis, washing,
and analysis by flow cytometry (Accuri C6).
Burzynski L.C., Morales-Maldonado A., Rodgers A., Kitt L.A., Humphry M., Figg N., Bennett M.R, & Clarke M.C. (2023). Thrombin-activated interleukin-1α drives atherogenesis, but also promotes vascular smooth muscle cell proliferation and collagen production. Cardiovascular Research, 119(12), 2179-2189.
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10

Immunophenotyping Murine Splenocytes

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Spleens were sieved (70 µm), before washing [phosphate-buffered saline (PBS); 350 g, 5 min], re-sieving (40 µm), RBC lysis (eBioscience), washing, and resuspension in FACS buffer (1% BSA, 0.05% NaN3, in PBS) or full RPMI 1640. Cells in FACS buffer were Fc blocked (1:100; BioLegend; 10 min, RT) before staining for T-cell activation with: anti-CD4 (1:800; eBioscience), anti-CD8 (1:100), anti-CD62L (1:80), anti-CD44 (1:400; all BioLegend; 20 min, RT); or for Tregs with: anti-CD4 (1:800), anti-CD25 (1:80; BioLegend; 20 min, RT), before washing, fixation, permeabilization (FOXP3 Fix/Perm; BioLegend), then anti-FOXP3 (1:20; BioLegend; 30 min, RT). Splenocytes in RPMI were treated ±ionomycin/phorbol 12-myristate 13-acetate/brefeldin A (1:500; BioLegend), incubated (5 h, 37°C), washed, resuspended in FACS buffer, Fc blocked, stained with anti-CD4 or anti-CD8 (20 min, RT), washed, fixed, permeabilized (BioLegend), then anti-IL-10, anti-IL-17, and anti-interferon γ (IFNγ; all 1:100; BioLegend; 30 min, RT). For neutrophil, monocyte or Ly6C+ cells, whole blood (ethylenediaminetetraacetic acid) was stained with anti-CD115 (1:100; eBioscience), anti-CD11b (1:800), anti-Ly6G (1:80; both BioLegend), anti-Ly6C (1:400; AbD Serotec, Hercules, CA), at RT for 30 min, before RBC lysis, washing, and analysis by flow cytometry (Accuri C6).
Burzynski L.C., Morales-Maldonado A., Rodgers A., Kitt L.A., Humphry M., Figg N., Bennett M.R, & Clarke M.C. (2023). Thrombin-activated interleukin-1α drives atherogenesis, but also promotes vascular smooth muscle cell proliferation and collagen production. Cardiovascular Research, 119(12), 2179-2189.
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