BM cells were flushed from mouse femurs with RPMI-1640 medium containing 2% fetal bovine serum and lysed using a lysis buffer (StemCell Technologies). Then 1 × 107 of the BM cells were stained with LSK, as described previously. The cells were incubated with anti-Annexin V-PE and anti-7AAD. The cells were tested using a flow cytometer (Thermo Fisher Scientific, USA), and the flow cytometry data were analyzed using FlowJo 7.6.1 (Tree Star, Franklin Lake, NJ, USA).
Lysis buffer
Manufactured by STEMCELL
Sourced in Canada
Lysis buffer is a solution used to disrupt and lyse cells, releasing their contents for further analysis or processing. It serves as a fundamental tool in various biological applications, such as protein extraction, nucleic acid purification, and cell biology studies. The buffer composition is designed to solubilize cell membranes and organelles, while preserving the integrity of the target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by STEMCELL (2 mentions)
Facsaria 2, by BD (1 mentions)
40 μm filter, by BD (1 mentions)
Percoll, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Nh4cl, by STEMCELL (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Aerrane, by Baxter (1 mentions)
Fetal bovine serum (fbs), by STEMCELL (1 mentions)
7 protocols using lysis buffer
1
Isolation and analysis of mouse bone marrow cells
2
Quantifying Reactive Oxygen Species in BM Cells
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BM cells were flushed from mouse femurs with RPMI-1640 medium containing 2% fetal bovine serum and lysed using a lysis buffer (StemCell Technologies). Then, 1 × 107 of the BM cells were stained with LSK, as described previously. Next, the cells were incubated with DCF (10 μM) for 30 min at 37 °C. After washing with PBS, the cells were analyzed using a flow cytometer (Thermo Fisher Scientific, USA).
3
Murine Splenic CD4+ T Cell Isolation
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Spleens were collected from mice four weeks after PBMC injection. Mouse spleens were prepared into single-cell suspensions then RBCs were lysed with 5–10 mL lysis buffer (Stem Cell Technology, Vancouver-Canada) for 10 min. Mouse splenocytes were enriched for human CD45+ cells using Miltenyi human CD45+ beads (Miltenyi Biotec, Cologne-Germany), according to the manufacturer’s recommendations. Then, human CD4+ Th cells were sorted to at least 95% purity from the human CD45+ enriched spleen using FACS Aria II (BD Biosciences, California-USA). Total RNA was isolated from sorted CD4+ Th cells using the Norgen kit per the manufacturer’s instructions (Norgen Biotek CORP, Ontario, BC, Canada).
4
Isolation of Immune Cells from Mouse Spinal Cord and Spleen
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Mice were deeply anesthetized with 5% isoflurane (Aerrane, Baxter) and transcardially perfused with 50 mL of cold phosphate-buffered saline, pH 7.4 (PBS, Sigma-Aldrich). Spinal cords were harvested at the EAE plateau phase [day post-immunization (dpi) 20 ± 3] and homogenized in PBS. Leukocytes were recovered at the 30:70% Percoll (Fisher Scientific) interface after gradient centrifugation as described in the literature [23 ] and were then counted with the Malassez chamber. Spleens were aseptically removed from naïve and MOG-immunized C57BL/6 mice at the peak of clinical score (≥ 3, dpi 15–18), as described previously [24 (link)], mechanically processed to obtain a splenocyte suspension by passing the cells through a 40-μm filter (Falcon). Erythrocytes were lysed in lysis buffer [0.15 M NH4Cl, 9 mM HKCO3, 0.5 M EDTA, pH 7.4 (Stemcell Technologies)], and the sterile splenocytes were resuspended in supplemented sterile PBS with 10% filtered and inactivated fetal bovine serum (FBS, Stemcell Technologies), 1% Penicillin/Streptomycin (Gibco), and 2.5% (v/v) HEPES (Fisher).
5
Neutrophil Quantification in BALF
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BALF was collected and centrifuged for 10 min at 300 g. The supernatants were stored at −80°C until detection, and the erythrocytes of the cell pellets were removed using lysis buffer (STEMCELL, Vancouver, Canada). After total cell counting, approximately 2 × 105 cells were loaded onto a slide by cytospin and stained with Giemsa stain to count the number of neutrophils.
6
Single-cell isolation from fetal liver and bone marrow
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Fetal livers from E15.5 embryos were dissociated by passing through a 21-gauge needle. The cells were passed through 40μM cell strainers to generate single-cell suspensions. Red blood cells were removed with lysis buffer (3% acetic acid in methylene blue) (Stem Cell Technologies). Bone marrow cells were flushed from hind limb bones through a 21-gauge needle into HBSS+ (Hanks balanced salt solution (Invitrogen,), 2% FBS, and 10 mM HEPES.
7
Single-cell isolation from fetal liver and bone marrow
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