Whole tissues were dissociated between glass slides and filtered through a 40-μm mesh. Spleen, lymph node and thymus samples were treated with Red Blood Cell Lysing Buffer (Sigma). Cells were then counted on a hemacytometer. Liver homogenate was suspended in Percoll (Sigma) to a final concentration of 30% Percoll. Liver homogenates were then centrifuged for 20 min at 2,000 r.p.m. in a Sorvall RTH-750 rotor. Lymphocytes were then collected from the buffy layer, washed once with FACS buffer, treated with Red Blood Cell Lysing Buffer (Sigma) and counted on a hemacytometer.
Red blood cell lysing buffer
Manufactured by Merck Group
Sourced in United States, Sao Tome and Principe
Red blood cell lysing buffer is a solution used in laboratory procedures to selectively disrupt and lyse red blood cells, while preserving other cell types. It is a common reagent used in various hematological and immunological analyses.
Automatically generated - may contain errors
Lab products found in correlation
70 μm cell strainer, by BD (7 mentions)
Dnase 1, by Roche (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Concanavalin a (cona), by Merck Group (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Collagenase d, by Roche (5 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (4 mentions)
Nylon cell strainer, by BD (4 mentions)
Dnase 1, by Merck Group (4 mentions)
Cytofix cytoperm kit, by BD (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Collagenase type 1, by Worthington (3 mentions)
Histopaque 1077, by Merck Group (3 mentions)
Ionomycin, by Merck Group (3 mentions)
2 mercaptoethanol, by Merck Group (3 mentions)
Rpmi 1640 medium, by Lonza (3 mentions)
Lipopolysaccharides (lps), by InvivoGen (3 mentions)
Mojosort mouse cd8a selection kit, by BioLegend (3 mentions)
159 protocols using red blood cell lysing buffer
1
Tissue Dissociation and Cell Isolation Protocol
2
Analysis of Tumor-Infiltrating Lymphocytes
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Splenocytes and intratumoral T lymphocytes were purified from OVA hyper-immune C57BL/6J mice challenged with MB49 bladder cancer cells treated or not with SFNs: specimens were minced and passed through a cell strainer to obtain a homogenous cell suspension. Then, lymphocytes were purified by centrifugation on a Ficoll gradient Lympholyte-H Cell Separation Media (Cedarlane, Burlington, Canada). After red blood cell lysis (red blood cell lysing buffer, Merck), splenocytes or tumor-infiltrating lymphocytes were seeded in 96 wells of round-bottomed plates (Corning, Somerville, Massachusetts, USA) in triplicate at 106 cells/well in a volume of 100 µL of culture medium (Gibco Life Technologies, Milan, Italy) in the presence or not of OVA (100 µg/mL) overnight. At the end of incubation, the cells were stained with Efluor V450 rat anti-mouse CD3 (Thermo Fisher catalog no 48-0032-82) and PE-Cyanine7 rat anti-mouse CD8 (BD catalog no 552877) antibodies, fixed and permeabilized by BD Cytofix Cytoperm (BD), and incubated with allophycocyanine rat anti-mouse interleukin (IL)-10 (Thermo Fisher catalog no 17-7101-82) and FITC rat anti-mouse interferon (IFN)-γ (Thermo Fisher catalog no 11-7311-82) antibodies. Cells were then analyzed by LSRFortessa X20 flow cytometer (BD).
3
Calyxoside B and C Cytokine Production
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C57BL/6 wild type mice were purchased from Clea Japan, Inc. Cd1d-deficientmice [16 (link)] were kindly provided by Dr. Luc van Kaer (Nashville, TN, USA). Mice were kept under specific pathogen-free conditions and were used at 8–16 wk of age. All experiments were in accordance with protocols approved by the Animal Care and Use Committee of Kanazawa University.
Spleen cells were collected and used after treatment with red blood cell lysing buffer (Merck KGaA). RPMI 1640 (Merck KGaA) containing 10% heat-inactivated fetal calf serum (Invitrogen), 5 × 10−5 M 2-mercaptoethanol, 2mM L-glutamine, 25 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (Mediatech, Manassas, VA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin was used as the culture medium. Spleen cells with single cell suspension were cultured with or without the 3:1 mixture of calyxoside B and calyxoside in the concentration of 105/100 μL. For the measurement of cytokine production, the culture supernatants were collected 72 h post incubation. Cytokines in culture supernatants were analyzed by cytometric bead array (BD Biosciences) according to the manufacturer’s protocol.
Spleen cells were collected and used after treatment with red blood cell lysing buffer (Merck KGaA). RPMI 1640 (Merck KGaA) containing 10% heat-inactivated fetal calf serum (Invitrogen), 5 × 10−5 M 2-mercaptoethanol, 2mM L-glutamine, 25 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (Mediatech, Manassas, VA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin was used as the culture medium. Spleen cells with single cell suspension were cultured with or without the 3:1 mixture of calyxoside B and calyxoside in the concentration of 105/100 μL. For the measurement of cytokine production, the culture supernatants were collected 72 h post incubation. Cytokines in culture supernatants were analyzed by cytometric bead array (BD Biosciences) according to the manufacturer’s protocol.
4
Isolation and Characterization of Porcine PMN
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Blood samples from two pigs (mixed-bred: Large White × Landrace) were collected using tubes containing EDTA. PMN were isolated using Ficoll-Paque Plus (GE Heathcare, Sweden) following manufacturer’s instructions, and contaminating erythrocytes were lysed using Red Blood Cell (RBC) Lysing Buffer (Sigma Aldrich, Brazil). The purity and cell identity were confirmed by flow cytometry taking into consideration the characteristics of the cells (FSC vs SSC) and the expression of the antigen recognized by monoclonal antibody (mAb) clone 6D10 isotype IgG2a (kindly provided by Dr. Javier Domínguez Juncal, Spain) followed by detection with biotin rat anti-mouse IgG2a (clone R19-15, BD Pharmingen™, USA) in the presence of streptavidin allophycocyanin conjugate (SA-APC) (BD Pharmingen™, USA). Cell viability was evaluated by propidium iodide (PI, Invitrogen, Brazil) and was consistently higher than 95%.
5
Immune Cell Depletion in Tumor-Bearing Mice
6
Thymocyte Isolation and Cryopreservation
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Thymocytes were collected from Minimum Dulbecco's Essential medium immediately after thymic tissue fragmentation. Contaminating red blood cells were eliminated by treatment with Red blood cell lysing buffer (from Sigma-Adrich, Milan, Italy), according to manufacturer's instruction. Thymocytes were frozen and stored in liquid nitrogen until used.
7
Cytokine Production Assay in Murine Splenocytes
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Mice were euthanatized at D28 and spleens collected and isolated via gentle extrusion of the tissue through a 50-μm-mesh nylon cell strainer (BD). Cells were resuspended in DMEM medium supplemented with 10% FCS, 2 mM L-glutamine, 50 U/mg penicillin and 50 U/mg streptomycin. Erythrocytes were lysed with red-blood-cell lysing buffer (Sigma-Aldrich). For stimulation experiments, 1 × 106 cells per well were stimulated for 48 h (37°C, 10% CO2) in DMEM medium in P24 plates in presence of PMA (phorbol 12-myristate 13-acetate) ionomycin cocktail 1× (eBioscience). Culture supernatant was frozen at -80°C until processing. Levels of the cytokines IL-6 (mouse IL-6 DuoSet ELISA, R&D), IL-17A, and IFN-γ (ELISA Development Kit, Mabtech) were determined using ELISA according manufacturer’s instructions.
8
Isolation of SCLC Tumor Cells
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Prior to processing, mouse SCLC and allograft tumors were decontaminated under the dissecting microscope by removing any normal and connective tissues. Then, tumors were transferred to a dry dish and minced into pieces with blades. The tissue was digested in Leibovitz’s medium (Invitrogen) with 2 mg/mL Collagenase Type I (Worthington), 2 mg/mL Elastase (Worthington), and 2 mg/mL DNase I (Worthington) at 37 °C for 45 min. The tissue was triturated with a pipet every 15 min of digestion until homogenous. The digestion was stopped with FBS (Invitrogen) to a final concentration of 20%. The cells were filtered with a 70 μm cell strainer (Falcon) and spun down at 5,000 r/min for 1 min. The cell pellet was resuspended in red blood cell lysing buffer (Sigma) for 3 min, spun down at 5,000 r/min for 1 min, and washed with 1 mL ice-cold Leibovitz’s medium with 10% FBS. Cells were resuspended in 1 mL ice-cold Leibovitz’s medium with 10% FBS and filtered with a cell strainer (20 μm). Dead cells were removed with Dead Cell Removal Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Live cells were collected for 10× Genomics library preparation.
9
Differentiation of Murine Bone Marrow Cells
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Female C57BL/6 mice, 6–8 weeks of age, were purchased from The Animal Laboratory of Chinese Academy of Sciences (Shanghai) and housed at the Central Animal Facility of Zhejiang University. All mice studies were approved by the Institutional Animal Care and Use Committee and were performed in accordance with the institutional guidelines. Bone marrow cells were isolated by flushing femurs and tibiae of mice with RPMI 1640 medium supplemented with 10% FCS, 10 mmol/L HEPES, 1 mmol/L sodium pyruvate, 2 mmol/L GlutaMAX (Life Technologies), 100 U/mL penicillin (Life Technologies), 100 mg/mL streptomycin (Life Technologies), and 0.1 mmol/L 2-ME. Red blood cells were lysed from bone marrow cell preparations using red blood cell lysing buffer (Sigma Aldrich). Bone marrow cells were cultivated for 5 days at a density of 1 × 106/mL supplemented with 100 ng/mL Flt-3L (PeproTech) for differentiation. The medium was changed once during cultures by replacing two-thirds of the medium with fresh cytokine-supplemented medium.
10
Single-cell Isolation from EAE Mouse
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Single cell suspensions were prepared by smashing brain and spinal cord tissues from EAE mice with a syringe and nylon mesh. The cells were collected by centrifugation and resuspended in 10 ml of 37% percoll (made in sterile PBS). After spinning 20 minutes at 4 °C (2000 × g), upper phase was removed and the mononuclear cells, contained in the pellet in the bottom of the tube, were collected. Cells were washed and treated with red blood cell lysing buffer (Sigma) to lyse erythrocytes and then resuspended in complete media.
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