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.
Buv805
Manufactured by BD
BUV805 is a fluorescent dye that can be used for flow cytometric analysis. It is designed to specifically bind to certain target molecules or cell surface markers, allowing researchers to identify and quantify the presence of those targets in a sample.
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)
Bb700, by BD (4 mentions)
Bv610, by BioLegend (4 mentions)
Buv563, by BD (4 mentions)
Collagenase type 4, by Worthington (4 mentions)
Facsymphony analyzer, by BD (3 mentions)
Facsdiva, by BD (2 mentions)
Celltrace violet fluorescent dye, by Thermo Fisher Scientific (1 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by BioLegend (1 mentions)
Facsymphony, by BD (1 mentions)
Foxp3 fix perm buffer kit, by Thermo Fisher Scientific (1 mentions)
Human ab serum, by Merck Group (1 mentions)
6 protocols using buv805
1
Profiling Immune Cell Populations in Inguinal Lymph Nodes
2
Profiling PBMC T-cell Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Participant PBMCs were thawed using a CryoThaw adaptor48 (link) into 10 mL of RPMI supplemented with 10% FBS. PBMCs were rested for approximately 4 h following thaw. Subsequently, 1e6 cells were labeled with cell trace violet fluorescent dye following the manufacturer’s instructions (Thermo Fisher) and cultured in 1 mL of RPMI supplemented with 10% FBS in FACS tubes and stimulated for five days with 1 mg/mL ancestral peptides (JPT Peptide Technologies) that have been resuspended in DMSO. Unstimulated samples were supplemented with equivalent volume DMSO. After five days, surface staining was performed for flow cytometry using Invitrogen Live/Dead Fixable Blue Dead Cell Stain and anti-human CD3 (BUV661, BD Biosciences), CD4 (BUV805, BD Biosciences), CD8α (BV510, BioLegend), CCR7 (BUV395, BD Biosciences) and CD45RA (APC-H7, BD Biosciences) antibodies. T-cell proliferation assays were performed on participants for which we had sufficient PBMC samples.
3
Optimizing Activated T Cell Expansion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total PBMCs were stimulated with α-CD3 antibody (OKT3, Biolegend) at 0.1 μg/ml for 2 days in complete RPMI 1640 media containing 2% heat-inactivated human AB serum (Sigma-Aldrich), washed, and rested in complete media for 5 days. On day 7, cells were harvested and 200 k cells were cultured in complete media containing only IL-2 mutein at titrating concentrations in a 96-well flat bottom plate. Five days later, cells were harvested and labeled with a viability dye (LIVE/DEAD Fixable Near-IR Dead Cell stain Kit, ThermoFisher Scientific) per manufacturer's protocol and stained for cell surface markers: CD3 (BUV805, BD Biosciences), CD4 (BUV395, BD Biosciences), CD8 (BV510, Biolegend), CD25 (BB515, BD Biosciences), and CTLA4 (a-CD152-PE/Cy7, Biolegend). Stained cells were fixed and permeabilized using the Foxp3 fix/perm buffer kit (eBioscience) and stained for Foxp3 (Alexa Fluor 647, Biolegend) and Ki67 (BV421, BD Biosciences). Samples were acquired on BD FACSymphony (BD Biosciences) and analyzed using FlowJo software.
The raw values of the percent Ki67-positive or Foxp3 and CTLA4 median fluorescence intensity were converted to percent response (% response) based on the values obtained from cells stimulated with wildtype IL-2 at 66.7 nM or media only (baseline). The equation that was used for conversion is as follows:
The raw values of the percent Ki67-positive or Foxp3 and CTLA4 median fluorescence intensity were converted to percent response (% response) based on the values obtained from cells stimulated with wildtype IL-2 at 66.7 nM or media only (baseline). The equation that was used for conversion is as follows:
4
Comprehensive Immune Cell Profiling of Vaccinated Mice
5
Comprehensive Immune Profiling of Iliac Lymph Nodes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
6
Comprehensive Immune Cell Profiling of Vaccinated Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!