Mice neutrophils were harvested and purified as previously described19 (link). In brief, bone marrow cells were gathered, added into Percoll gradient consisting of 52%, 65%, 78% Percoll layers (GE, Boston, MA, USA), and centrifuged at 2500 ×g for 30 min at room temperature. The cells between 65% and 78% layers were harvested, and red blood cells were lysed with red blood cells lysis buffer (BD, New York, NY, USA).
Red blood cells lysis buffer
Manufactured by BD
Sourced in United States
Red blood cells lysis buffer is a solution used to lyse or break down red blood cells in a sample, typically to isolate other cellular components for analysis. It facilitates the removal of red blood cells, which can interfere with downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Percoll, by GE Healthcare (1 mentions)
Percoll gradient, by GE Healthcare (1 mentions)
Aurora flow cytometer, by Cytek (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Fortessa, by BD (1 mentions)
Fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
4 protocols using red blood cells lysis buffer
1
Isolation of Neutrophils from Mouse Bone Marrow
2
Spleen Cell Isolation and Viability
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For cell and cytokine flow cytometry analysis, the larger fragment of the spleen was weighed, then macerated in a cell strainer at 70 μm (BD Falcon) coupled to a 50 mL conical tube filled with phosphate-buffered saline (PBS). The cell suspension was centrifuged at 1,600 rpm/5 min/4°C. The cell-containing pellet was incubated in 2 mL of Red Blood Cells Lysis Buffer (BD Biosciences) for 2 min/37°C then centrifugated at 1,400 rpm/5 min/4°C. The cells were resuspended in 1 mL PBS and then the cell viability was assessed by trypan blue staining.
3
Isolation of Single Cell Suspension from Rat Tumor Tissue
4
Comprehensive Flow Cytometry Analysis of cTFH Cells
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Whole-blood aliquots were incubated with a mixture of monclonal Abs: anti-CD3, -CXCR5, -CD4, -CXCR3, -CD25 (BD, San Jose, CA) and -CD45RO, -PD1, -CD62L, CD127 (eBioscience, San Diego, CA), in the dark for 30 minutes at room temperature. Samples were then incubated with red blood cells lysis buffer (BD) for 10 minutes at room temperature, washed twice, and fixed with fixation/permeabilization buffer (eBioscience) for 40 minutes at 4 °C. For T regulatory cells (TREG) and Ki67 detection, cells were further permeabilized with permeabilization buffer (eBioscience) and incubated in the dark for 30 minutes at 4 °C with anti-FOXP3 and anti-Ki67 (eBioscience), washed twice, and immediately acquired. All events were acquired using a Fortessa (BD) cytometer and analyzed with FlowJo software (Tree Star, Ashland, OR). In addition, we applied t-distributed Stochastic Neighbor Embedding algorithm (FlowJo) and integrated the fluorescence intensity of phenotypic markers into a consensus map to visualize the overall differences of cTFH cell subset composition measured by cell cluster density.
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