Single cell suspensions were prepared from spleens by mechanical homogenization between glass slides, filtered through nylon mesh, and RBC lysed with ACK lysis buffer (Thermo Fisher) for 5 minutes at 37°C. Cells were then washed and resuspended in cell sorting buffer (HBSS + 5% FBS + 5 mM EDTA). Cells were stained for 20–45 minutes at 4°C. The following antibodies/reagents were used for cell sorting experiments: anti-CD19 FITC (clone 6D5 Biolegend), anti-B220 AF700 (clone RA3–6B2 Biolegend), anti-CD95 PE-Cy7 (clone Jo.2 BD Biosciences), anti-GL7 ef450 (clone GL7 eBiosciences), NP-PE (Biosearch Technologies), anti-IgG1 APC (clone RMG1–1 Biolegend), LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies). Cells were washed twice and resuspended in 500µl cell sorting buffer. Bulk GC B cells (300,000 – 500,000 cells) from pooled (n = 4/group) spleens of NK cell depleted or control treated animals or from spleens of individual mice (n = 4–8/group) receiving either NK-depleting antibody or isotype control were sorted on a BD FacsAria cell sorter into 1.5 mL Eppendorf tubes containing 200 µL of cell sorting buffer.
Np pe
Manufactured by Biosearch Technologies
Sourced in United Kingdom
NP-PE is a laboratory equipment product offered by Biosearch Technologies. It is a compact and versatile instrument designed for performing various analytical and research tasks in a laboratory setting. The core function of NP-PE is to facilitate the detection and analysis of specific molecules or compounds, enabling researchers and scientists to conduct their experiments and investigations effectively.
Automatically generated - may contain errors
Lab products found in correlation
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (2 mentions)
Anti cd19 fitc clone, by BioLegend (2 mentions)
Anti b220 af700 clone ra3 6b2, by BioLegend (2 mentions)
Anti cd95 pe cy7 clone jo 2, by BD (2 mentions)
Anti gl7 ef450 clone, by Thermo Fisher Scientific (2 mentions)
Anti igg1 apc, by BioLegend (2 mentions)
Np2 bsa, by Biosearch Technologies (2 mentions)
Facsaria 3, by BD (2 mentions)
Live dead fixable viability dye, by Thermo Fisher Scientific (1 mentions)
Lsr fortessa 16 color cell analyzer, by BD (1 mentions)
Fixable viability dye, by Thermo Fisher Scientific (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Moflo astrio, by Beckman Coulter (1 mentions)
Fortessa, by BD (1 mentions)
Cd19 microbeads, by Miltenyi Biotec (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Streptavidin apc, by BD (1 mentions)
Streptavidin pe, by BD (1 mentions)
Facs calibur 2, by BD (1 mentions)
11 protocols using np pe
1
Isolation of Germinal Center B Cells
2
Flow Cytometric Analysis of Immune Cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immediately after suspension, cells were stained for flow cytometric analysis with LIVE/DEAD fixable viability dye (Life Technologies, Carlsbad, CA, United States) and the following antibodies: CD90.2, CD44, CD8a, CD4, CD62L, CD11a, CXCR5, PD1, CD69, B220, CD19, CD23, CD21/35, GL7, IgM, IgD, CD138, and CD93. NP-PE (Biosearch Technologies, Teddington, Middlesex, United Kingdom) was utilized to detect NP-specific cells. Full antibody details are available in Supplementary Table S1 . All flow cytometric analysis was performed on a BD LSR Fortessa 16-color cell analyzer and analyzed using FlowJo software version 10 (BD Biosciences, San Jose, CA, United States). Gating Strategy for T cells is shown in Supplementary Figure S2 and for B cells is shown in Supplementary Figure S3 .
3
Characterization of NP-specific B cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells from the spleen were isolated, and single-cell suspensions were stained with a fixable viability dye (Life Technologies), then pretreated with unlabeled anti-CD16/CD32 (Fc Block; BD Pharmingen). Cells were then stained in FACS buffer (2.5% fetal calf serum plus 0.1% sodium azide) with the following mixtures of directly conjugated antibodies, purchased from BioLegend, unless otherwise noted: anti-mouse CD19, IgD, IgM, CD21/35, CD23 (BD Pharmingen), GL-7, CD38, CD4, CD8a, GR-1, F4/80, MHCII, and CD138. Identification of NP-specific B cells was performed by staining with NP-PE, with a conjugation ratio of 23NP/PE (N-5070; Biosearch Technologies). For proliferation studies, cells were labeled with CellTrace violet (Life Technologies).
For P-S6 staining, cells were fixed with 4% paraformaldehyde at 37°C for 10 min, washed in FACS buffer, and fixed in 90% methanol for 20 min on ice. Cells were washed in FACS buffer and stained with anti–P-S6–biotin for 1 h at room temperature. Cells were washed in FACS buffer, and fluorochrome-conjugated streptavidin was added for 15 min at 4°C.
Samples were analyzed using a Fortessa (BD Biosciences) flow cytometer, and flow-cytometric sorting was performed using the MoFlo Astrios (Beckman Coulter) at the CHOP flow cytometry core facility. Data analysis was done using FlowJo (Treestar).
For P-S6 staining, cells were fixed with 4% paraformaldehyde at 37°C for 10 min, washed in FACS buffer, and fixed in 90% methanol for 20 min on ice. Cells were washed in FACS buffer and stained with anti–P-S6–biotin for 1 h at room temperature. Cells were washed in FACS buffer, and fluorochrome-conjugated streptavidin was added for 15 min at 4°C.
Samples were analyzed using a Fortessa (BD Biosciences) flow cytometer, and flow-cytometric sorting was performed using the MoFlo Astrios (Beckman Coulter) at the CHOP flow cytometry core facility. Data analysis was done using FlowJo (Treestar).
4
Comprehensive B Cell Immunophenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were blocked with α-CD16/32 clone 93 (BD Pharmingen) and stained with the following fluorochrome-coupled antibodies purchased from either eBioscience or BD Pharmingen: α-AA4.1 clone AA4.1, α-B220 clone RA3-6B2, α-BP1 clone BP-1, α-CD5 clone 53-7.3, α-CD16/32 clone 93, α-CD19 clone 1D3, α-CD21 clone 7G6, α-CD23 clone B3B4, α-CD38 clone 90/CD38, α-CD43 clone S7, α-CD138 clone 281-2, α-HSA clone M1/69, α-Igα clone 24C2.5, α-λ5 clone LM34, α−IgG1 clone R3-3, α-GL7 clone GL7, α-Igκ clone 187.1, NP-PE (Biosearch Technologies), streptavidin-PE (#554061, BD Pharmingen), streptavidin-APC (#554067, BD Pharmingen), α-IgMµ-APC (#115-175-075, Jackson ImmunoResearch Europe), and IgMµ-PE (#1020-09S, SouthernBiotech). The stained cells were then subjected to FACS analysis on a special-order LSR II equipped with a UV, a violet, and a yellow-green laser (BD Biosciences) or sorted with either a BD Influx or a BD FACSAria III (BD Biosciences). For intracellular FACS staining, the BD Cytofix/Cytoperm kit (BD Biosciences) was employed, and, in the case of surface µHCs, the surface was blocked with 10 µg of unlabeled α-IgMµ (#31172, Thermo Fisher Scientific). MACS purification of splenic CD19+ cells was performed using CD19 Microbeads (Miltenyi) according to the manufacturer’s protocol.
5
Isolation of Germinal Center B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single cell suspensions were prepared from spleens by mechanical homogenization between glass slides, filtered through nylon mesh, and RBC lysed with ACK lysis buffer (Thermo Fisher) for 5 minutes at 37°C. Cells were then washed and resuspended in cell sorting buffer (HBSS + 5% FBS + 5 mM EDTA). Cells were stained for 20–45 minutes at 4°C. The following antibodies/reagents were used for cell sorting experiments: anti-CD19 FITC (clone 6D5 Biolegend), anti-B220 AF700 (clone RA3–6B2 Biolegend), anti-CD95 PE-Cy7 (clone Jo.2 BD Biosciences), anti-GL7 ef450 (clone GL7 eBiosciences), NP-PE (Biosearch Technologies), anti-IgG1 APC (clone RMG1–1 Biolegend), LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies). Cells were washed twice and resuspended in 500µl cell sorting buffer. Bulk GC B cells (300,000 – 500,000 cells) from pooled (n = 4/group) spleens of NK cell depleted or control treated animals or from spleens of individual mice (n = 4–8/group) receiving either NK-depleting antibody or isotype control were sorted on a BD FacsAria cell sorter into 1.5 mL Eppendorf tubes containing 200 µL of cell sorting buffer.
6
Multiparameter Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with FACS buffer (HBSS containing 0.2% BSA and 0.05% Azide), treated with anti-FcR (24G2), and then stained with specific antibodies. Anti–mouse CD4, CD8, PD-1, CXCR5, B220, CD19, Fas, GL7, CD38, CD11c, B7.1, B7.2, CD40, CXCR4, CD83, TCR-Vα2, TCR-Vβ5, CD45.1, and CD45.2 antibodies were purchased from BD Biosciences. NP-PE was purchased from Biosearch Technologies. Propidium iodide was purchased from Sigma-Aldrich. For intracellular cytokine staining, splenic CD4+ T cells were stimulated with PMA and ionomycin for 2 h, and cells were fixed and permeabilized with the BD Fix/Perm kit (BD Biosciences) according to the manufacturer’s instructions and then stained with anti–IFN-γ (BD Biosciences) and isotype control antibody for 30 min. Data were collected with a FACS Calibur II, FACS LSR II, FACS Fortessa, or FACS Aria III flow cytometer (BD Biosciences) and analyzed with FlowJo software.
7
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescently-conjugated or biotin-conjugated antibodies to B220, CD5, CD19, CD21, CD23, CD69, CD86, CD93 (AA4.1), CD95 (Fas), CD138, CXCR4, IgA, IgM, IgM[a], IgM[b], IgD, IgD[a], IgG1[a], IgG1[b], Igκ, Igλ, GL-7, MHC Class II, and fluorescently-conjugated streptavidin were from Biolegend, eBiosciences, BD Biosciences, Tonbo, or Life Technologies. NP-PE was from Biosearch Technologies. 100 nm Rhodamine PtC liposomes were from FormuMax. Antibodies for intra-cellular staining, pErk Ab (clone 194g2) and pS6 Ab (2F9), were from Cell Signaling Technologies, and Nur77 Ab (clone 12.14) conjugated to PE was from eBioscience. Goat anti-mouse IgM F(ab’)2 was from Jackson Immunoresearch. Goat anti-mouse Igκ and goat F(ab’)2 anti-mouse Igκ were from Southern Biotech. Anti-IgD was from MD Biosciences. CXCR4 ligand (CXCL12/hSDF-1α) was from Peprotech. LPS (Cat. L8274) was from Sigma. CpG DNA (ODN 1826 Biotin; Cat. tlrl-1826b) and Pam3CSK4 (Cat. tlrl-pms) were from InvivoGen.
8
Murine B Cell Phenotyping and Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorophore-labeled anti-B220 (RA3-6B2) and anti-FAS (Jo2) antibodies were from BD; anti-CD138 (281-2), anti-CD21/35 (CR2/CR1), and anti-IgD (11-26c2a) antibodies were from BioLegend; anti-GL7 (GL7), anti-CD38 (90), and anti-rabbit IgG antibodies were from Invitrogen; rabbit anti-cCasp3 and the matching isotype control antibodies were from Cell Signaling; and anti-GFP antibody was from Abcam. The fixable viability dye, zombie yellow, was from BioLegend. NP18-OVA, NP30-Ficoll, NP2-BSA, NP-PE, and 4-hydroxy-3-iodo-5-nitrophenylacetyl (NIP)-BSA-biotin were from Biosearch Technologies. NP-APC conjugates were made by allowing NP-Osu (Biosearch Technologies) and APC (BioLegend) to react for 4 h at room temperature in a buffer containing 0.1 M NaHCO3 and 0.15 M NaCl2 (pH 8), at a molar ratio of 20:1. Dextran (200 kD, 31398) and Dextran-FITC (2,000 kD, FD2000S) were from Sigma-Aldrich.
9
Multiparametric Flow Cytometry Analysis
10
Multiparameter Flow Cytometry Analysis
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!