External ears were collected from Bcl3-KO and WT mice; minced and digested with collagenase type I (200U/mL; Worthington) and dispase II (0.9U/mL; Roche) in a shaking water bath at 37°C for 2h. Dissociated cells were filtered through a 70 µm cell strainer and centrifuged at 1000r for 5 min. Pelleted cells were resuspended for 1 min in red blood cell lysing buffer (Solarbio) to remove erythrocyte contamination and were centrifuged at 1000r for 5 min. The isolated cells were plated in 100mm Petri dishes (P0) in Dulbecco’s modified Eagle’s medium (DMEM; Corning) supplemented with 1% antibiotic solution and 10% fetal bovine serum (FBS; Gibco). Subconfluent primary cultures were detached (0.25% trypsin, 2.21mM EDTA; Corning), replated in complete medium, and subcultured a density of 5×104 cells/mL in six-well culture plates.
Red blood cell lysing buffer
Manufactured by Solarbio
Red blood cell lysing buffer is a solution used to lyse or break down red blood cells in a biological sample. It is a key component in the sample preparation process, allowing for the isolation and analysis of other cell types or analytes of interest.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Corning (1 mentions)
Dispase 2, by Roche (1 mentions)
Trypsin, by Corning (1 mentions)
Collagenase type 1, by Worthington (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Corning (1 mentions)
Porcine peripheral blood lymphocyte separation kit, by Solarbio (1 mentions)
Minimacs starting kit, by Miltenyi Biotec (1 mentions)
Mouse igg1 kappa isotype control, by Thermo Fisher Scientific (1 mentions)
Goat anti pig igm, by Bio-Rad (1 mentions)
Cd80 b7 1 monoclonal antibody, by Thermo Fisher Scientific (1 mentions)
Anti fitc microbeads, by Miltenyi Biotec (1 mentions)
Recombinant mouse m csf, by Thermo Fisher Scientific (1 mentions)
100 μm basket filter, by BD (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
4 protocols using red blood cell lysing buffer
1
Isolation and Culture of Murine Ear Cells
2
Porcine Peripheral Blood Lymphocyte Isolation and Characterization
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Porcine peripheral blood lymphocyte separation kit and red blood cell lysing buffer were purchased from Solarbio Science & Technology (Beijing) Co., Ltd. Enzyme-Linked Immunosorbent Assay (ELISA) kits were purchased from Dongge Biological (Beijing) Co., Ltd. Mouse Anti-Porcine CD21 IgG was purchased from Southern Biotech (Alabama). Anti-FITC MicroBeads and MiniMACS Starting kits were purchased from Miltenyi Biotec (Bergisch-Gladbach). Anti-CD54/ICAM1 antibody was purchased from Arigo (Taiwan). Mouse IgG1 kappa Isotype Control was purchased from Invitrogen (Waltham). Goat anti-Pig IgM was purchased from Bio-Rad (California). CD80 (B7-1) Monoclonal Antibody was purchased from eBioscience (California). Mouse anti-Pig-SLA Class II DR and mouse anti-Mouse MHC Class I were purchased from AbD Serotec (Kidlington).
3
Differentiation of Murine Bone Marrow Macrophages
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Femora and tibiae of C57BL/6 mice were harvested and the bone marrow cells from all bones were flushed out. Then after centrifuging cells for 5 min at 500 ×g, erythrocytes were eliminated using red blood cell lysing buffer (Cat#: R1010, Solarbio). The remaining cells were seeded in plates and incubated in complete DMEM medium overnight, and then replaced with fresh medium with 10 ng/ml of recombinant mouse M-CSF (Cat#: AF-315-02, PeproTech), for 2 days.
4
Isolating Immune Cells from Murine Samples
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Cells from peripheral blood, bronchoalveolar lavage (BAL) and lung tissues were isolated as previously described 11 . Briefly, mice were anesthetized with isoflurane, and peripheral blood was exhaustively collected from the abdominal vein. Following exhaustive BAL with PBS, lung lobes were collected, cut into small pieces and digested in collagenase type I (Thermo Fisher) for 1 h at 37 °C in a shaker incubator. Lung single-cell suspensions were obtained by crushing the digested lung tissues through a 100-μm basket filter (BD Biosciences). Following erythrocyte lysis of peripheral blood and lung cells by incubation with red blood cell lysing buffer (SolarBio), cells were washed with PBS and resuspended in desired medium for further experiments.
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