Draining iliac lymph nodes from BNT162b2 immunized mice, draining inguinal lymph nodes from YF-17D immunized mice, along with whole lung or spleen, were harvested and digested with 1 mg/mL collagenase type IV (Worthington) for 20 min at 37 °C, followed by smashing with a 100 μm strainer to make a single-cell suspension. Red blood cells from the lung and spleen were lysed before staining. Single-cell samples were then stained with Zombie UV™ (1:300, BUV496; BioLegend #423107), anti-Ly6C (1:500, BV780; BioLegend #128041), anti-Ly6G (1:400, APC-Cy7; BioLegend #127624), anti-CD19 (1:100, BB700; BD #566411), anti-CD3 (1:100, BB700; BD #742175), anti-MHCII (1:400, AF700; eBioscience #56-5321-82), anti-CD11b (1:300, BV650; BioLegend #101239), anti-CD11c (1:400, BV421; BioLegend #117330), anti-CD86 (1:300, A647; BioLegend #105020), anti-Siglec-F (1:400, PE-CF594; BD #562757), anti-CD45 (1:200, BV610; BioLegend #103140), anti-CD169 (1:200, PE-Cy7; BioLegend #142412), anti-PDCA-1 (1:200, BUV563; BD #749275), anti-CD8a (1:200, BUV805; BD #612898), anti-CD103 (1:100, PE; eBioscience #12-1031-82), anti-NK1.1 (1:200, BV510; BioLegend #108738), and anti-F4/80 (1:100, BUV737; BD #749283). Data was collected on a BD FACSymphony analyzer with BD FACSDiva (v8.0.1).
Bb700
Manufactured by BD
Sourced in United States
The BB700 is a laboratory centrifuge designed for general-purpose applications. It features a brushless motor and can achieve a maximum speed of 4,500 RPM. The centrifuge has a capacity of up to 6 x 50 mL tubes and can accommodate a range of rotor options. The BB700 is suitable for a variety of sample preparation and separation tasks in research and clinical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Apc cy7, by BioLegend (4 mentions)
Buv496, by BioLegend (4 mentions)
Bv780, by BioLegend (4 mentions)
Bv610, by BioLegend (4 mentions)
Buv563, by BD (4 mentions)
Buv805, by BD (4 mentions)
Collagenase type 4, by Worthington (4 mentions)
Facsymphony analyzer, by BD (3 mentions)
Facsdiva, by BD (2 mentions)
Fitc conjugated mouse igg1, by BD (1 mentions)
Fitc conjugated mouse igg2a, by BD (1 mentions)
Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions)
Accuri c6 plus, by BD (1 mentions)
Deoxyribonuclease, by Merck Group (1 mentions)
6 protocols using bb700
1
Comprehensive Immune Cell Profiling of Vaccinated Mice
2
Comprehensive Immune Profiling of Iliac Lymph Nodes
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Iliac lymph nodes were harvested at day 1 post-vaccination. LNs were treated with 5 mg/ml collagenase type IV (Worthington) for 20 min at 37 °C and smashed against a 100μm strainer to make a single-cell suspension. Cells were then stained with Zombie UV™ (BUV496; Biolegend # 423108, 1:250 dilution), anti-CD64 (A488; Biolegend # 139316, 1:100 dilution), anti-Ly6C (BV780; Biolegend # 128041, 1:500 dilution), anti-Ly6G (APC-Cy7; Biolegend# 127624, 1:400 dilution), anti-CD19 (BB700; BD # 566411, 1:200 dilution), anti-CD3 (BB700; BD #742175, 1:200 dilution), anti-MHCII (AF700; eBioscience #56-5321-82, 1:400 dilution), anti-CD11b (BV650; Biolegend #101239, 1:300 dilution), anti-CD11c (BV421; Biolegend #117330, 1:400 dilution), anti-CD86 (A647; Biolegend #105020, 1:300 dilution), anti-Siglec-F (PE-CF594; BD #562757, 1:400 dilution), anti-CD24 (BUV395; BD #744471, 1:200 dilution), anti-CD45 (BV610; Biolegend #103140, 1:300 dilution), anti-CD169 (PE-Cy7; Biolegend #142412, 1:200 dilution), anti-PDCA-1 (BUV563; BD #749275, 1:200 dilution), anti-CD8a (BUV805; BD #612898, 1:200 dilution), anti-CD103 (PE; eBioscience #12-1031-82, 1:100 dilution), anti-NK1.1 (BV510; Biolegend #108738, 1:200 dilution), anti-F4/80 (BUV737; BD #749283, 1:100 dilution). Cells were analyzed with the BD FACSymphony analyzer at Stanford HIMC core.
3
Comprehensive Immune Cell Profiling of Vaccinated Mice
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Draining iliac lymph nodes from BNT162b2 immunized mice, draining inguinal lymph nodes from YF-17D immunized mice, along with whole lung or spleen, were harvested and digested with 1 mg/mL collagenase type IV (Worthington) for 20 min at 37 °C, followed by smashing with a 100 μm strainer to make a single-cell suspension. Red blood cells from the lung and spleen were lysed before staining. Single-cell samples were then stained with Zombie UV™ (1:300, BUV496; BioLegend #423107), anti-Ly6C (1:500, BV780; BioLegend #128041), anti-Ly6G (1:400, APC-Cy7; BioLegend #127624), anti-CD19 (1:100, BB700; BD #566411), anti-CD3 (1:100, BB700; BD #742175), anti-MHCII (1:400, AF700; eBioscience #56-5321-82), anti-CD11b (1:300, BV650; BioLegend #101239), anti-CD11c (1:400, BV421; BioLegend #117330), anti-CD86 (1:300, A647; BioLegend #105020), anti-Siglec-F (1:400, PE-CF594; BD #562757), anti-CD45 (1:200, BV610; BioLegend #103140), anti-CD169 (1:200, PE-Cy7; BioLegend #142412), anti-PDCA-1 (1:200, BUV563; BD #749275), anti-CD8a (1:200, BUV805; BD #612898), anti-CD103 (1:100, PE; eBioscience #12-1031-82), anti-NK1.1 (1:200, BV510; BioLegend #108738), and anti-F4/80 (1:100, BUV737; BD #749283). Data was collected on a BD FACSymphony analyzer with BD FACSDiva (v8.0.1).
4
Profiling Immune Cell Populations in Inguinal Lymph Nodes
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Draining inguinal lymph nodes from immunized mice were collected and treated as previously described (47 (link), 48 (link)). Briefly, the tissues were treated with collagenase type IV (Worthington) at a concentration of 1 mg/mL for 20 min at a temperature of 37 °C, and then passed through a 100-μm strainer to obtain a single-cell suspension. The resulting single-cell samples were stained with a range of markers including Zombie UV (BUV496, BioLegend 423107), anti-Ly6C (BV780, BioLegend 128041), anti-Ly6G (APC-Cy7, BioLegend 127624), anti-CD19 (BUV395, BD 563557), anti-CD3 (BB700, BD742175), anti-MHCII (AF700, eBioscience 56-5321-82), anti-CD11b (BV650, BioLegend 101239), anti-CD11c (BV421, BioLegend 117330), anti-CD86 (A647, BioLegend 105020), anti-Siglec-F (PE-CF594, BD 562757), anti-CD45 (BV610, BioLegend 103140), anti-CD169 (PE-Cy7, BioLegend 142412), anti-PDCA-1 (BUV563, BD 749275), anti-CD8a (BUV805, BD 612898), anti-CD103 (PE, eBioscience 12-1031-82), anti-NK1.1 (BV510, BioLegend 108738), and anti-F4/80 (BUV737, BD 749283). The cells were analyzed using the BD FACSymphony analyzer located at the Stanford Shared Fluorescence-activated cell sorting (FACS) Facility.
5
Integrin Expression Analysis by Flow Cytometry
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Cells were incubated with FITC-conjugated anti-CD49d(α4) (557457, BD) and anti-CD61(β3) (561909, BD) and BB700-conjugated anti-CD49e(α5) (745884, BD). FITC-conjugated mouse IgG2a (553456, BD), FITC-conjugated mouse IgG1 (550616, BD), and BB700 hamster IgG1 were used as negative control, respectively. The detailed method was followed in accordance with the manufacturer’s instructions. Cells were analyzed with an EVOS® FL Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA) and a BD Accuri™ C6 Plus (BD Biosciences, Franklin Lakes, NJ, USA).
6
Leukocyte Isolation and Characterization
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The rats (three rats per group) were anesthetized and transcardial perfusion with PBS. Different brain regions were separated into 70 μm strainer, homogenized by the needle bladder, and collected into tubes. Deoxyribonuclease (D4527‐10KU, Sigma, 50,000 kU/mL) was added to the tubes and then centrifuged at 4°C, 1500 rpm for 10 min. The sediment was resuspended with 30% Percoll. The suspension was poured slowly down the inner wall of the tubes with 70% Percoll so that the different density of Percoll clearly separated. The cocktail was centrifuged gradient 30 min at 600 G (20°C) with no brake. Then collected 2.0 ± 0.5 mL of the interphase between 30% and 70% Percoll into a clean 15 mL conical tube and added isopycnic HBSS. The samples were centrifuged at 4°C 2000 rpm for 5 min and the sediment was the target leukocytes. The cells were then incubated with CD marker antibodies (CD45, 1:200, BB700, OX‐1, BD Pharmingen, United States; CD11b, 1:200, FITC, WT.5, BD Pharmingen, United States; CD86, 1:200, BV650, 24F, BD Pharmingen, United States) for 30 min at 4°C in darkness and were analyzed by flow cytometry (Bechman, MoFlo Astrios EQS).
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