BM cells of femur and tibia of C57BL/6 (Shanghai Laboratory Animal Center, Shanghai, China) and enhanced green fluorescent protein (EGFP+) mice (Model Animal Research Center of Nanjing University, Nanjing, China) at 6–8 wk of age were passed through a 70 μm cell strainer and centrifuged for 5 min at 400 ×g. After RBC lysis (Beyotime, Shanghai, China), cells were incubated with recombinant murine IL‐4 (1 ng/ml) (R&D Systems, Minneapolis, MN, USA) and GM‐CSF (10 ng/ml) (R&D Systems) for 48 h. Suspended cells were gently removed and adherent cells were further cultured. On day 6 of culture, the CD11C+ BMDCs isolated by negative selection with anti‐mouse CD11C magnetic beads (Miltenyi, Teterow, Germany) were collected and further pretreated with or without hemin (7.5 μmol/L, Sigma‐Aldrich, St Louis, MO, USA) for 2 h to induce HO‐1 expression and then stimulated with house dust mite (HDM; 50 μg/ml, Greer lab, Boston, MA, USA) or treated with PBS as a control for 24 h.
Rbc lysis
Manufactured by Beyotime
Sourced in China
The RBC lysis product is a reagent used to lyse, or break down, red blood cells (RBCs) in a sample. It is a core laboratory tool that facilitates the isolation and analysis of other cellular components, such as white blood cells, from a blood sample.
Automatically generated - may contain errors
2 protocols using rbc lysis
1
Differentiation and Activation of BMDCs
2
Neutrophil Extraction from Whole Blood
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Whole blood from BU patients and HCs was collected in heparin tubes. Within 3 h after sample collection, we first diluted 4 mL blood with the same amount of phosphate buffer saline (PBS) solution, then carefully layered it over Ficoll (TBD) in a 15 mL tube, and centrifuged it with Ficoll at 350× g for 20 min at 4 °C. The neutrophil layer was collected by quickly pouring the contents into a new 15 mL tube. After neutrophil collection, red blood cell (RBC) lysis (Beyotime) was used to obtain pure neutrophils. The cell suspension was washed twice with PBS and stored for subsequent processing.
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