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Fluorophore conjugated antibodies

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Fluorophore-conjugated antibodies are laboratory reagents that consist of antibodies covalently linked to fluorescent dyes, known as fluorophores. These conjugated antibodies are used to detect and quantify specific target molecules in various laboratory techniques, such as flow cytometry, immunofluorescence microscopy, and Western blotting.

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Lab products found in correlation

Fc block, by BD (3 mentions) Cytofix cytoperm, by BD (3 mentions) Lsrfortessa, by BD (2 mentions) Flow cytometry, by BD (2 mentions) Ter119, by BD (2 mentions) Accuri c6 flow cytometer, by BD (2 mentions) Fixable viability dye, by Thermo Fisher Scientific (1 mentions) Facs lsr 2, by BD (1 mentions) Fortessa, by BD (1 mentions) Fortessa flow cytometer, by BD (1 mentions) Balb c mice, by Nara Biotech (1 mentions) Sf900ii media, by Thermo Fisher Scientific (1 mentions) C6 analysis software, by BD (1 mentions) Lsrii cytometer, by BD (1 mentions) Sh800s cell sorter, by Sony (1 mentions) Transcription factor staining kit, by Thermo Fisher Scientific (1 mentions) Flica poly caspase assay kit, by ImmunoChemistry Technologies (1 mentions) Aria 2, by BD (1 mentions) Facs lsr 2 flow cytometer, by BD (1 mentions) Facsdiva software, by BD (1 mentions)

27 protocols using fluorophore conjugated antibodies

1

Multiparametric flow cytometry analysis

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After harvesting or isolation, mICcl2cells, BMDCs, cLP cells and mLN cells were washed and Fc-receptors were blocked for 15 min at 4°C. Staining with fixable viability dyes (Thermo Fisher Scientific) for 15 min at 4°C was applied for live-dead discrimination. For intracellular staining, cells were fixed and permeabilized using Cytofix/Cytoperm (BD Biosciences) according to manufacturer’s instructions, washed and resuspended in PBS/1% FCS/0.1% saponin. For intracellular staining and cell surface staining, cells were labeled for 30 min at 4°C with fluorophore-conjugated antibodies (all BD, if not stated otherwise) and washed twice. Flow cytometric analyses were performed on a FACS LSRII (BD Biosciences). Data were analyzed using the FlowJo software (Tree Star Inc., USA). Antibodies: CD11c (HL3)-APC, CD11c (HL3)-PE-Cy7, CD4 (RM4-5)-BV605, CD45 (30-F11)-APC-Cy7, CD45R (RA3-6B2)-PE, CD64 (X54-5/7.1)-PE, Foxp3 (MF23)-AF647, Foxp3 (MF23)-BV421, GATA3 (L50-823)-PE-Cy7, IFNγ (XMG1.2)-PE-Cy7, IFNγ (XMG1.2)-APC, IFNγ (XMG1.2)-FITC, IκBζ (LK2NAP)-PerCP-EF710 (Thermo Fisher Scientific), IL-10 (JES5-16E3)-BV510, IL-10 (JES5-16E3)-FITC (BioLegend), IL-17A (TC11-18H10)-PE, IL-17A (TC11-18H10)-APC-Cy7, IL-4 (11B11)-PE, LY6G/C (RB6-8C5)-PE, I-A/I-E (MHC II) (AF6-120.1)-APC, I-A/I-E (MHC II) (AF6-120.1)-BV421, RORγt (Q31-378)-BV421, and T-bet (4B10)-BV421 (BioLegend).
Michaelis L., Treß M., Löw H.C., Klees J., Klameth C., Lange A., Grießhammer A., Schäfer A., Menz S., Steimle A., Schulze-Osthoff K, & Frick J.S. (2021). Gut Commensal-Induced IκBζ Expression in Dendritic Cells Influences the Th17 Response. Frontiers in Immunology, 11, 612336.
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2

Multicolor Flow Cytometric Analysis of T Cell Subsets

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Single cell suspensions were prepared from thymus. Cells were incubated with various combinations of fluorophore-conjugated antibodies against the following cell-surface markers (BD Biosciences) at room temperature for 20 min: Thy1.2, CD28, c-Kit, CD4, CD8, CD44, CD25, CD69, TCRβ, IL-7Rα, HSA, CD62L, and TCR Vα2. Immunolabeled cells were analyzed by flow cytometry.
Intracellular TCRβ staining was performed as described previously (26 (link)). Briefly, cells were incubated with fluorophore-conjugated antibodies to cell-surface markers (anti-CD4, anti-CD8, anti-Thy1.2, anti-CD44, and anti-CD25, anti-CD28), as well as with a saturating amount of anti-TCRβ to block cell-surface TCRβ. Cells were then fixed at room temperature for 20 minutes with 1% paraformaldehyde. After washing in PBS, the cells were incubated with 10 mM glycine for 10 minutes to quench autofluorescence. Cells were then permeabilized using 0.5% saponin, 5% FCS, and 10 mM Hepes (pH 7.4) at room temperature for 10 minutes and stained with fluorophore-conjugated antibody to TCRβ at room temperature for 45 minutes. Intracellular staining of Bcl2 was carried out similarly, except that the Bcl2 antibody was revealed with secondary fluorophore-conjugated goat anti–mouse IgG (Jackson ImmunoResearch).
Zhang S., Konstantinidis D.G., Yang J.Q., Mizukawa B., Kalim K., Lang R.A., Kalfa T.A., Yi Z, & Guo F. (2014). Gene targeting RhoA reveals its essential role in coordinating mitochondrial function and thymocyte development. Journal of immunology (Baltimore, Md. : 1950), 193(12), 5973-5982.
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3

Quantifying T Cell Subset Changes in SIV Infection

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Peripheral blood mononuclear cells were isolated from peripheral blood samples as previously described (Crakes et al., 2019 (link)). Changes in the distribution of T cell subsets and CD4+ T cell depletion were assessed in SIV infected animals compared to uninfected controls. Multi-color immunophenotyping was performed on BD Fortessa with a minimum of 300,000 events collected. Fluorophore conjugated antibodies were obtained from BD Biosciences and used for the detection of CD45RA (MEM-56)–PE-TexasRed, CD3 (SP34)-allophycocyanin (APC)-Cy7, CD4 (OKT4)-Pacific Blue and CD8 (3B5)-APC-Cy5.5. Cells were evaluated using BD Fortessa flow cytometer. Data was analyzed using FlowJo software (v10.4.1; Tree Star, Inc., Ashland, OR) using double discrimination and live amine dyes (Invitrogen).
Hu S., Buser E., Arredondo J., Relyea D., Santos Rocha C, & Dandekar S. (2022). Altered Expression of ACE2 and Co-receptors of SARS-CoV-2 in the Gut Mucosa of the SIV Model of HIV/AIDS. Frontiers in Microbiology, 13, 879152.
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4

Plasmodium berghei Infection Model in BALB/c Mice

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BALB/c mice (six-week-old, female) were purchased from NARA Biotech (Seoul, Korea). Plasmodium berghei ANKA strain 2.34 was serially maintained in mice through intraperitoneal passaging. Recombinant baculovirus (rBV) and VLPs were generated using Spodoptera frugiperda Sf9 cells. The Sf9 cells were cultured in spinner flasks with SF900II media purchased from Invitrogen (Carlsbad, USA). P. berghei whole antigens were prepared following the method previously described [15 (link),16 (link),23 (link),24 (link)]. Blood samples were collected from P. brerghei-infected mice at 8 weeks post-infection via retro-orbital plexus puncture for serum acquisition. Monoclonal anti-M1 antibody and horseradish peroxidase (HRP)-conjugated secondary mouse IgG antibodies were purchased from Abcam (Cambridge, UK) and Southern Biotech (Birmingham, AL, USA), respectively. Fluorophore-conjugated antibodies were purchased from BD Bioscience (CA, USA) and used to perform flow cytometry.
Lee S.H., Chu K.B., Kang H.J, & Quan F.S. (2022). Protection and Alleviated Inflammation Induced by Virus-like Particle Vaccines Containing Plasmodium berghei MSP-8, MSP-9 and RAP1. Vaccines, 10(2), 203.
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5

Analyzing Immune Cell Dynamics in Spleen and Lung

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The activity of immune cells in the spleen and lung was analyzed by flow cytometry as described previously [21 (link)]. Percentages of T cells (CD4+, CD8+) and B cell (germinal center) from splenocytes and lung cells of mice were analyzed by flow cytometry 4 days post-challenge as described previously [6 ,22 (link)]. Splenocytes and lung cells (1×106 cell/mL) in staining buffer (2% bovine serum albumin and 0.1% sodium azide in 0.1 M PBS) were incubated at 4°C for 15 min with Fc Block (BD Biosciences, CA, USA). For surface antigen staining, cells were incubated with fluorophore-conjugated antibodies (CD3e, CD4, CD8, B220, GL7; BD Biosciences) at 4°C for 30 min. Splenocytes and lung cells were washed with staining buffer and fixed with 4% paraformaldehyde for 30 min at 4°C before acquisition using a BD Accuri C6 Flow Cytometer (BD Biosciences). Data were analyzed using C6 Analysis software (BD Biosciences).
Kang H.J., Chu K.B., Lee D.H., Lee S.H., Park B.R., Kim M.C., Kang S.M, & Quan F.S. (2019). Influenza M2 virus-like particle vaccination enhances protection in combination with avian influenza HA VLPs. PLoS ONE, 14(6), e0216871.
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6

Multiparameter Flow Cytometry for Immune Phenotyping

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Single-cell suspensions were generated from the various organs and stained with the indicated fluorophore-conjugated antibodies (BD Biosciences, eBioscience, BioLegend, and Tonbo). MHC class I tetramers were generated by conjugating Kb/SIINFEKL (Ova), Kb/SSPPMFRV (M38), or Db/HGIRNASFI (M45) monomers (NIH Tetramer Facility) to streptavidin-phycoerythrin (BD Biosciences/Life Technologies). The M3/fMIGWII tetramer was kindly provided by Dr. Eric Pamer (MSKCC). Flow cytometry was performed on a LSR II cytometer (BD), cells were sorted on an Aria (BD), and data were analyzed with the FlowJo software (Tree Star).
Johnson L.R., Weizman O.E., Rapp M., Way S.S, & Sun J.C. (2016). Epitope-specific vaccination limits clonal expansion of heterologous naïve T cells during viral challenge. Cell reports, 17(3), 636-644.
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7

Isolation and Activation of CD8+ T cells

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Cell sorting was performed on a Sony SH800S cell sorter (Sony Biotechnology). Staining was performed in PBS supplemented with 1% FBS. Sorting and cells collection for ex vivo naïve CD8+ T cells and 24 h TCR activated CD8+ T cell with or without inhibitor was performed in RPMI 1640 containing glutamine, supplemented with 1% FBS. Sorting and cell collection for FOXO1-GFP KO and WT CTL was performed in RPMI 1640 containing glutamine, supplemented with 10% FBS.
Fluorophore-conjugated antibodies were obtained from BD Biosciences, eBioscience or Biolegend.
Spinelli L., Marchingo J.M., Nomura A., Damasio M.P, & Cantrell D.A. (2021). Phosphoinositide 3-Kinase p110 Delta Differentially Restrains and Directs Naïve Versus Effector CD8+ T Cell Transcriptional Programs. Frontiers in Immunology, 12, 691997.
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8

Single-cell Immunophenotyping and Apoptosis Analysis

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Cell surface staining of single-cell suspensions from the indicated organs was performed using fluorophore-conjugated antibodies (BD Biosciences, eBioscience, BioLegend, Tonbo Biosciences, and R&D Systems). For intracellular cytokine staining, cells were fixed and permeabilized with the transcription factor staining kit (eBioscience). Apoptosis was evaluated by caspase activity staining using the carboxy-fluorescein FLICA Poly Caspase Assay kit (ImmunoChemistry Technologies). NK cell proliferation was analyzed by labeling cells with 5 μM CTV (Invitrogen) before transfer into recipient mice, and CTV labeling was performed according to the manufacturer’s protocol. Flow cytometry and cell sorting were performed on the LSR II and Aria II cytometers (BD Biosciences), respectively. The data were analyzed using FlowJo software (Tree Star).
Rapp M., Lau C.M., Adams N.M., Weizman O.E., O’Sullivan T.E., Geary C.D, & Sun J.C. (2017). Core-binding factor β and Runx transcription factors promote adaptive natural killer cell responses. Science immunology, 2(18), eaan3796.
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9

Multiparametric Flow Cytometry Analysis

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Splenocytes were stained with fluorophore-conjugated antibodies purchased from either BD Biosciences (San Jose, CA), eBioscience (San Diego, CA), or Cell Signaling Technologies (Danvers, MA): B220 (RA3-6B2), Bcl-6 (K112-91), CD4 (RM4-5), CD8a (53-6.7), CD25 (7D4), CD95 (Jo2), CD122 (TM-B1), Foxp3 (FJK-16s), IgM (II/41), Ly49C/F/I/H (14B11), PD-1 (J43), pSMAD2/3 (D27F4), and pSMAD1/5/8 (D5B10). Samples were acquired on a BD LSR Fortessa Flow Cytometer and analyzed by using FlowJo software (TreeStar, Ashland, OR).
Stocks B.T., Wilhelm A.J., Wilson C.S., Marshall A.F., Putnam N.E., Major A.S, & Moore D.J. (2015). Lupus-Prone Mice Resist Immune Regulation and Transplant Tolerance Induction. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 16(1), 334-341.
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10

Macrophage Phenotype Characterization

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The levels of surface markers of differentiation (CD11b or CD14) and polarization (HLA-DR and CD80 for M1 or CD206 and CD163 for M2a and c phenotypes) were assessed in THP-1 cells/PBMs and TDMs/MDMs of different phenotypes. Selected markers of M1 (HLA-DR) and M2 (CD206) phenotypes were analysed in MDMs treated with 500 nM NMU-9 (for 24 h) using immunofluorescence staining and flow cytometry. Macrophages were detached on ice with cold 0.5% BSA in PBS with 2 mM EDTA, resuspended in PBS/0.5% BSA/2 mM EDTA and stained with fluorophore-conjugated antibodies (all from BD Biosciences, Franklin Lakes, NJ, USA, described in Additional file 2: Table S1) for 30 min at RT in the dark. A washing step with PBS containing 2 mM EDTA was performed. The FACS analysis was performed using a FACS LSR II BD flow cytometer (Becton Dickinson) equipped with BD FACS Diva Software. The results were analysed using FlowJo™ 10.7.1 software (BD).
Przygodzka P., Soboska K., Sochacka E., Pacholczyk M., Braun M., Kassassir H., Papiewska-Pająk I., Kielbik M, & Boncela J. (2022). Neuromedin U secreted by colorectal cancer cells promotes a tumour-supporting microenvironment. Cell Communication and Signaling : CCS, 20, 193.
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