Total TAMs (CD45+/Gr1−/CD11b+/DAPI−) were sorted from GBM xenografts through the fluorescence-activated cell sorting (FACS). Briefly, GBM xenografts were resected and mechanically dissociated and then digested in HBSS buffer containing DNase I (10 μg/mL, Sigma Aldrich, 10104159001) and Liberase (25 μg/mL, Roche, 5401020001) for 45 min at 37°C, and mixed by pipetting every 10 min. After digestion, cell suspensions were passed through a 70 μm filter, washed with cold PBS, and spun down (800 rpm) for 10 min at 4°C. Red blood cells in the samples were removed with the specific lysis buffer (BioLegend, 420301). The dissociated cells were re-suspended in RPMI 1640 medium and blocked with rat IgG (Santa Cruz, sc-2026) for 15 minutes before staining with specific antibodies for sorting. Antibodies used for FACS include: anti-CD45 (Biolegend, 103127, Clone 30-F11), anti-Gr1 (Biolegend, 108405, Clone RB6-8C5), and anti-CD11b (Biolegend, 101207, clone M1/70). The sorted TAMs (CD45+/Gr1−/CD11b+/DAPI−) were then used for RNA-seq analyses or the flow cytometric analyses to quantify macrophage phagocytosis of glioma cells in vivo as described below.
Specific lysis buffer
Manufactured by BioLegend
Sourced in United States
The Specific lysis buffer is a reagent designed for the effective lysis and extraction of cellular components, including proteins and nucleic acids, from biological samples. It is a concentrated buffer solution formulated to disrupt cell membranes and release the contents within. The buffer composition may vary depending on the specific application and can be used in various research and diagnostic procedures.
Automatically generated - may contain errors
Lab products found in correlation
Clone 30 f11, by BioLegend (2 mentions)
Clone rb6 8c5, by BioLegend (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Liberase, by Roche (2 mentions)
40 μm cell strainer, by BD (1 mentions)
Cellxvivo mouse th2 cell differentiation kit, by R&D Systems (1 mentions)
Balb c mice, by Japan SLC (1 mentions)
Macs na ve cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
3 protocols using specific lysis buffer
1
Isolation and Characterization of Tumor-Associated Macrophages from Glioblastoma Xenografts
2
Isolation and Characterization of Tumor-Associated Macrophages from Glioblastoma Xenografts
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Total TAMs (CD45+/Gr1−/CD11b+/DAPI−) were sorted from GBM xenografts through the fluorescence-activated cell sorting (FACS). Briefly, GBM xenografts were resected and mechanically dissociated and then digested in HBSS buffer containing DNase I (10 μg/mL, Sigma Aldrich, 10104159001) and Liberase (25 μg/mL, Roche, 5401020001) for 45 min at 37°C, and mixed by pipetting every 10 min. After digestion, cell suspensions were passed through a 70 μm filter, washed with cold PBS, and spun down (800 rpm) for 10 min at 4°C. Red blood cells in the samples were removed with the specific lysis buffer (BioLegend, 420301). The dissociated cells were re-suspended in RPMI 1640 medium and blocked with rat IgG (Santa Cruz, sc-2026) for 15 minutes before staining with specific antibodies for sorting. Antibodies used for FACS include: anti-CD45 (Biolegend, 103127, Clone 30-F11), anti-Gr1 (Biolegend, 108405, Clone RB6-8C5), and anti-CD11b (Biolegend, 101207, clone M1/70). The sorted TAMs (CD45+/Gr1−/CD11b+/DAPI−) were then used for RNA-seq analyses or the flow cytometric analyses to quantify macrophage phagocytosis of glioma cells in vivo as described below.
3
Differentiation of Th2 Cells from Naïve CD4+ T Cells
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Five-week-old male BALB/c mice (weighing 18–20 g) were obtained from Japan SLC, Inc. (Shizuoka, Japan) and maintained in a controlled, specific pathogen-free environment (temperature, 24 °C ± 2 °C; humidity, 50% ± 5%; and light/dark cycle, 12/12 h with food and water provided ad libitum). Spleen tissues were passed through 40-μm cell strainers (Falcon, New York, NY, USA), and red blood cells were removed using a specific lysis buffer (BioLegend, San Diego, CA, USA). Naïve CD4+ T cells were isolated from the splenocytes using the Naïve CD4+ T Cell Isolation Kit (MACS; Miltenyi Biotec Inc., Auburn, CA, USA) and were differentiated into Th2 cells using the CellXVivo Mouse Th2 Cell Differentiation Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturers’ instructions. The animal experiments for the differentiation of Th2 cells from naïve CD4+ T cells were approved by the institutional animal care committee of Korea Institute of Oriental Medicine (KIOM: #20-070).
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