The following mAbs purchased from BioLegend (San Diego, CA) were used: N418 (anti-murine CD11c); 2.4G2 (“Fc block”, anti-murine CD16/CD32); 30-F11 (anti-murine CD45); C/23 (anti-murine CD40); 16-10A1 (anti-murine CD80); GL1 (anti-murine CD86); AF6-120.1 (anti-murine I-Ab); 10F.9G2 (anti-murine PD-L1); TY25 (anti-murine PD-L2); 6d5 (anti-murine CD19); 1A8 (anti-murine Ly-6G); 145-2C11 (anti-murine CD3ε); BM8 (anti-murine F4/80); NLDC-145 (anti-murine CD205); C068C2 (anti-murine CD206); XMG1.2 (anti-IFNγ); TC11-18H10.1 (anti-IL-17A); and TRFK4 (anti-IL-5). The mAb, AL-21 (anti-murine Ly-6C); M1/70 (anti-murine CD11b) and ebio13A (anti-IL-13) were purchased from BD Biosciences (San Diego, CA). The mAb 2f8 (anti-murine CD204) was purchased from AbD Serotec. Monoclonal Abs were primarily conjugated with Alexa Fluor 700, FITC, PE, PE-Cy7, PerCP-Cy5.5, allophycocyanin (APC), APC-Cy7, Brilliant Violet 421, or Pacific blue. Isotype-matched control mAbs (BioLegend) were tested simultaneously in all experiments.
Isotype matched control mabs
Manufactured by BioLegend
Sourced in United States
Isotype-matched control monoclonal antibodies (mAbs) are laboratory reagents used in flow cytometry and other immunoassays to serve as negative controls. These control mAbs are designed to match the isotype of the primary antibody being used in the experiment, but lack specificity for the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Af6 120, by BioLegend (2 mentions)
M1 70, by BD (2 mentions)
Al 21, by BD (2 mentions)
Tc11 18h10, by BioLegend (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Collagenase d, by Roche (1 mentions)
Symphony a3 analyzer, by BD (1 mentions)
Facscalibur system, by BD (1 mentions)
4 protocols using isotype matched control mabs
1
Comprehensive Murine Immune Cell Profiling
2
Isolation and Flow Cytometry Analysis of Immune Cells
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The LLNs were teased with 26 G needles and digested in 1 ml of 2.5 mg/ml collagenase D (Roche) solution in 1X RPMI at 37°C for 30 min. Digestion was stopped with 100 μl EDTA (100 mM). The cells were homogenized with glass Pasteur pipettes and then filtered through a 70-μm nylon filter. Single-cell suspensions were collected and centrifuged at 300 g for 5 min.
mAbs and isotype-matched control mAbs purchased from BioLegend were used for flow cytometry staining: PE-conjugated to CD26, Vα2, and SiglecF; PerCP-Cy5.5–conjugated to CD64, XCR1, and CD4; PE-Cy7–conjugated to CD11c and CD45.1; BUV395-conjugated to CD11b; BUV805-conjugated to CD8a, FITC-conjugated to F4/80, Ly6C, and CD103; allophycocyanin-conjugated to CD88 and CD64; APC-Cy7–conjugated to Ly6C, Ly6G, CD45, and CD11c; BV421-conjugated with Ly6G; and BV510-conjugated to MHCII and CD45.2. The viability dye DAPI (#D9542; Sigma-Aldrich) was added immediately before each sample acquisition on a BD Symphony A3 analyzer (BD Biosciences). Data were analyzed using FlowJo (Tree Star). Antigen-specific antibodies and isotype controls were obtained from BioLegend, eBioscience, and BD Biosciences.
mAbs and isotype-matched control mAbs purchased from BioLegend were used for flow cytometry staining: PE-conjugated to CD26, Vα2, and SiglecF; PerCP-Cy5.5–conjugated to CD64, XCR1, and CD4; PE-Cy7–conjugated to CD11c and CD45.1; BUV395-conjugated to CD11b; BUV805-conjugated to CD8a, FITC-conjugated to F4/80, Ly6C, and CD103; allophycocyanin-conjugated to CD88 and CD64; APC-Cy7–conjugated to Ly6C, Ly6G, CD45, and CD11c; BV421-conjugated with Ly6G; and BV510-conjugated to MHCII and CD45.2. The viability dye DAPI (#D9542; Sigma-Aldrich) was added immediately before each sample acquisition on a BD Symphony A3 analyzer (BD Biosciences). Data were analyzed using FlowJo (Tree Star). Antigen-specific antibodies and isotype controls were obtained from BioLegend, eBioscience, and BD Biosciences.
3
Comprehensive Immune Cell Profiling
4
Evaluation of NK Cell Phenotype in DLBCL
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NK cells were seeded into 96-well plates at a density
of 1×105 cells per well in DMEM/F12 culture medium
without FBS/AB serum. Then, NK cells were treated
with or without 20 µg (21 (link)) plasma-derived exosomes of
refractory/relapsed or responsive patients with DLBCL at
37°C, 5% CO2 for 24 hours. NK cells were harvested and
the percentage of NK cells expressing CD16, NKG2D and
CD69 was determined by flow-cytometry, followed by
comparing them in DLBCL patients and healthy donors.
The following anti-human monoclonal antibodies were
used for flow-cytometry: CD16 monoclonal antibody
(B73.1, PE); CD314 (NKG2D) monoclonal antibody
(1D11, PE); CD56 (NCAM) monoclonal antibody
(CMSSB, PE); CD3 monoclonal antibody (OKT3,
FITC) and CD69 monoclonal antibody (FN50, FITC;
all purchased from eBioscience™). The cells were also
stained with their corresponding isotype-matched control
mAbs (Bio-Legend, USA). All samples were analyzed
using the BD FACS Calibur system (Becton Dickinson
Co., USA). Flowing Software version 2.5.1 (TerhoPerttu,
Finland) was used for data acquisition and analysis.
of 1×105 cells per well in DMEM/F12 culture medium
without FBS/AB serum. Then, NK cells were treated
with or without 20 µg (21 (link)) plasma-derived exosomes of
refractory/relapsed or responsive patients with DLBCL at
37°C, 5% CO2 for 24 hours. NK cells were harvested and
the percentage of NK cells expressing CD16, NKG2D and
CD69 was determined by flow-cytometry, followed by
comparing them in DLBCL patients and healthy donors.
The following anti-human monoclonal antibodies were
used for flow-cytometry: CD16 monoclonal antibody
(B73.1, PE); CD314 (NKG2D) monoclonal antibody
(1D11, PE); CD56 (NCAM) monoclonal antibody
(CMSSB, PE); CD3 monoclonal antibody (OKT3,
FITC) and CD69 monoclonal antibody (FN50, FITC;
all purchased from eBioscience™). The cells were also
stained with their corresponding isotype-matched control
mAbs (Bio-Legend, USA). All samples were analyzed
using the BD FACS Calibur system (Becton Dickinson
Co., USA). Flowing Software version 2.5.1 (TerhoPerttu,
Finland) was used for data acquisition and analysis.
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