Apc fire 750

Manufactured by BioLegend

The APC/Fire 750 is a fluorescent dye that can be used in flow cytometry applications. It has excitation and emission spectra that make it suitable for detection on instruments with appropriate lasers and filters.

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Lab products found in correlation

Percp efluor 710, by Thermo Fisher Scientific (2 mentions) Mouse fc block, by BD (1 mentions) Fortessa x20 flow cytometer, by BD (1 mentions) 7 aad, by BioLegend (1 mentions) Fjk 16, by Thermo Fisher Scientific (1 mentions) Apc cy7, by BioLegend (1 mentions) Pe efluor 610, by Thermo Fisher Scientific (1 mentions) Apc efluor 780, by Thermo Fisher Scientific (1 mentions) H57 597, by BioLegend (1 mentions) Dta 1, by Thermo Fisher Scientific (1 mentions) Ra3 6b2, by Thermo Fisher Scientific (1 mentions) U29 93, by BD (1 mentions) Super bright 645, by Thermo Fisher Scientific (1 mentions) Rmst2 2, by Thermo Fisher Scientific (1 mentions) Foxp3 transcription factor staining buffer, by Thermo Fisher Scientific (1 mentions) Rm4 5, by BioLegend (1 mentions) Trustain fcx, by BioLegend (1 mentions) M1 70, by BioLegend (1 mentions) Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions) Fluorescein isothiocyanate (fitc), by BioLegend (1 mentions)

Close spelling variants of this product

HLA-DR-APC/Fire™ 750 (6 citations) Anti-CD4-APC/Fire 750 (4 citations) Anti-CD8 APC/Fire 750 (4 citations) CD19-APC-Fire 750 (3 citations) Anti-CD3 APC/Fire 750 (3 citations) Ly6G-APC/Fire 750 (3 citations) Anti-human-CD45-APC/Fire 750 (2 citations) Anti-Vδ2-APC/Fire 750 (2 citations) Anti-DC-SIGN APC/Fire 750 (2 citations) KLRG1-APC/Fire 750 (2 citations)

7 protocols using apc fire 750

Podoplanin and Apoptosis Analysis

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5555 melanoma cells were analysed following doxycycline induction of podoplanin. Cells were incubated with purified rat anti-mouse CD16/CD32 (Mouse BD Fc Block™, 0.5μL/test, BD Biosciences, 553142) as per supplier's instructions. Cells were stained with hamster anti-Podoplanin-PE (clone 8.1.1 (RUO), 0.5μL/test, BD Biosciences, 566390) diluted in PBS supplemented with 5% BSA and 5mM EDTA for 30 min in dark at 4°C. Cells were then stained using the Annexin V (APC/Fire™ 750, Biolegend, 640953) and 7-AAD (Biolegend, 420404) as per supplier's instructions. Stained cells were analysed using the Fortessa X-20 flow cytometer (BD Biosciences). All flow cytometry data was analysed using FlowJo Software version 10 (BD Biosciences).

de Winde C.M., George S.L., Crosas-Molist E., Hari-Gupta Y., Arp A.B., Benjamin A.C., Millward L.J., Makris S., Carver A., Imperatore V., Martínez V.G., Sanz-Moreno V, & Acton S.E. (2021). Podoplanin drives dedifferentiation and amoeboid invasion of melanoma. iScience, 24(9), 102976.

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Multiparameter Flow Cytometry Assay

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Antibodies against murine CD4 (RM4-5, AF700, BV650), CD8 (53-6.7, APC/Fire 750, BV785), CCR2 (SA203G11, BV421), CD11c (N418, APCCy7, Pe/Cy5), CD206 (C068C2, APC), CD11b (M1/70, BV421), F4/80 (BM8, BV510, BV711, FITC), TCRβ (H57-597, BV605), and NK1.1 (PK136, BV650) were purchased from BioLegend. Antibodies against CD4 (GK1.5, BUV395) and ST2 (U29-93, BV480) were purchased from BD Biosciences. Antibodies against B220 (RA3-6B2, AF488), CD11c (N418, e450, PE-Cy5.5), CD25 (PC61.5, PE-Cy7), CD4 (GK1.5, APC-eFluor 780, Super Bright 645), CD45 (30-F11, Pacific Orange), Foxp3 (FJK-16s, APC, e450, FITC), GITR (DTA-1, Super Bright 600), IL-10 (JES5-16E3, PE), KLRG1 (2F1, APC, APC-eFluor 780, PE-eFluor 610), NK1.1 (PK136, PE), and ST2 (RMST2-2, PE, PerCP-eFluor710) were purchased from eBioscience, Thermo Fisher Scientific. Viability dyes (e506, near IR, UV) were purchased from Invitrogen, Thermo Fisher Scientific.
Extracellular flow staining was performed in 2% FBS-PBS in the presence of BioLegend’s TruStain FcX for 30 minutes at 4°C. Fixation, permeabilization, and intracellular staining were performed using eBioscience’s (Thermo Fisher Scientific) Foxp3/transcription factor staining buffer set per manufacturer’s directions. Flow data were collected using a Cytek Aurora or BD Biosciences LSR and analyzed using FlowJo.

Beppu L.Y., Mooli R.G., Qu X., Marrero G.J., Finley C.A., Fooks A.N., Mullen Z.P., Frias AB J.r., Sipula I., Xie B., Helfrich K.E., Watkins S.C., Poholek A.C., Ramakrishnan S.K., Jurczak M.J, & D’Cruz L.M. (2021). Tregs facilitate obesity and insulin resistance via a Blimp-1/IL-10 axis. JCI Insight, 6(3), e140644.

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Comprehensive Immune Cell Analysis of Bronchoalveolar Lavage

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Bronchoalveolar lavage pellets were stained with antibodies and analyzed on a Beckman Coulter “Cytoflex” 13-color cytometer (Beckman Coulter, Brea, CA, United States). Antibodies used include CD16/32 (TruStain FcX PLUS, Biolegend, Catalog 156603), CD45 (APC-Fire 750, Biolegend, Catalog 147714), Ly-6G (Alexa Fluor 488, Biolegend, Catalog 127626), CD11a/CD18 (PE, Biolegend, Catalog 141006), CD11b/CD18 (APC, Biolegend, Catalog 101212), CD62L (BV421, Biolegend, 104435), CD49d (PE, Biolegend 103608, Catalog 103608), CD44 (AAPC, Biolegend, Catalog 103012), and CD162 (BV421, BD Biosciences, Catalog 562807). We identified live/dead cell ratios by using propidium iodide (PI, Fisher Scientific Catalog BDB556463).

Cagle L.A., Linderholm A.L., Franzi L.M., Last J.A., Simon S.I., Kenyon N.J, & Harper R.W. (2022). Early mechanisms of neutrophil activation and transmigration in acute lung injury. Frontiers in Physiology, 13, 1059686.

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Characterization of CAR T Cell Phenotype and Function

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To evaluate the in vivo CAR T cells, whole blood and mononuclear cell preparations from tissue biopsies were stained with antibodies labeled with fluorochromes for cytometric analysis. All samples (100 μL whole blood or 10⁶ small lymphocytes from tissue samples or cultured CAR T cells) were initially stained with anti-EGFR (Hu1: Biotin, R&D Systems), and Live/Dead Fixable Aqua Dead Cell Stain Kit (Thermo Fisher) for 30 min. After washing, the samples were stained with surface antibodies 30 min: CD45 (D058-1283: BUV395, BD Biosciences), anti-CD8a (SK1: BUV737, BD Biosciences), streptavidin (BV421, BD Biosciences), anti-CXCR5 (MU5UBEE: Super Bright 600, eBioscience), anti-CCR7 (150503: BV711, BD Biosciences), anti-CD4 (L200: BV786, BD Biosciences), anti-CD95 (DX2: PE, BioLegend), anti-CD28 (CD28.2: PE-DAZZ, BioLegend), anti-PD-1 (J105, PerCP-eFluor710, eBioscience), anti-CCR5 (3A9: APC, BD Biosciences), anti-CD3 (SP34-2, Alexa Fluor700, BD Biosciences), and anti-CD20 (2H7: APC/Fire750, BioLegend). Intracellular staining with anti-Ki67 (B56: FITC, BD Biosciences) was performed for 45 min after lyse/Fix (BD Biosciences) and permeabilizations. Polychromatic (8–14 parameter) flow-cytometric analysis was performed on a LSR II BD instrument as previously described.⁵¹ (link) List mode multiparameter data files were analyzed using the FlowJo software program (Tree Star, v.9.9.6).

Haeseleer F., Fukazawa Y., Park H., Varco-Merth B., Rust B.J., Smedley J.V., Eichholz K., Peterson C.W., Mason R., Kiem H.P., Roederer M., Picker L.J., Okoye A.A, & Corey L. (2021). Immune inactivation of anti-simian immunodeficiency virus chimeric antigen receptor T cells in rhesus macaques. Molecular Therapy. Methods & Clinical Development, 22, 304-319.

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Flow Cytometric Identification of NK Cells and Neutrophils

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Whole blood (50 μL) was transferred to flow cytometry tubes and incubated for 30 minutes in the dark at room temperature with 50 μL antibody master mix prepared in PBS containing 0.5% BSA (flow buffer). Red blood cells were then lysed by two rounds of incubation with 2 mL of High-Yield Lyse buffer (Life Technologies) followed by centrifugation at 500 × g. Cells were resuspended in flow buffer and analyzed by flow cytometry using either LSRFor-tessa or FACSCanto instruments (BD Biosciences). Antibodies against CD3ε (Brilliant Violet 421, #100335), CD49b/DX5 (FITC, #108905), and CD11b (APC/Fire 750, #101261) were obtained from BioLegend and used according to the supplier’s instructions. Natural killer (NK) cells were identified as the CD3ε-negative, CD49b/DX5– positive population. Neutrophils/monocytes were identified as the CD11b-positive population.

Kumar D., Rahman H., Tyagi E., Liu T., Li C., Lu R., Lum D., Holmen S.L., Maschek J.A., Cox J.E., VanBrocklin M.W, & Grossman D. (2018). Aspirin Suppresses PGE2 and Activates AMP Kinase to Inhibit Melanoma Cell Motility, Pigmentation, and Selective Tumor Growth In Vivo. Cancer prevention research (Philadelphia, Pa.), 11(10), 629-642.

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Multiparameter Flow Cytometry of Immune Cells

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A total of 1 × 10⁶ mononuclear cells suspended in PBS were stained using incubation with 1 µg Fc Block (anti-CD16/32, Tonbo Biosciences) for 15 min at 4 °C. Cells were then stained with the following antibodies: CD45 (APC/Fire 750-Biolegend Clone: 13/2.3), CD11c (APC-Biolegend, Clone: N418) MHCII (PerCp Cy5.5-Biolegend, Clone: M5/114.15.2), CD88 (PE, Biolegend, Clone: 20/70), CD317 (BV421, Biolegend, Clone: 927), CD45.1 (FITC-Biolegend Clone: A20), and CD45.2 (BV605, Biolegend, Clone: 104) for 45 min at 4 °C. Events were recorded via BD FACS LSR Fortessa (The Moody Foundation Flow Cytometry Facility, UT Southwestern), equipped with Diva acquisition software (BD Bioscience). For FACS sorting, BD FACSARIA-II SORP (The Moody Foundation Flow Cytometry Facility, UT Southwestern) was utilized. Cells were gated according to morphology based on their side scatter (SSC) vs. forward scatter (FSC) distributions. Doublets were excluded (FSC-A vs. FSC-H and SSC-A vs. SSC-H, where A corresponds to area and H corresponds to height). Gating for FACS sorting consisted of singlet CD45⁺MHCII⁺CD88⁺CD317⁺ CD45.1⁺ cells or CD45⁺MHCII⁺CD88⁺CD317⁺CD45.2⁺ cells. In each sample, a minimum of 50 × 10³ live events were recorded. FlowJo software (BD Bioscience) was used for data analysis.

Manouchehri N., Salinas V.H., Hussain R.Z, & Stüve O. (2023). Distinctive transcriptomic and epigenomic signatures of bone marrow-derived myeloid cells and microglia in CNS autoimmunity. Proceedings of the National Academy of Sciences of the United States of America, 120(6), e2212696120.

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Flow Cytometry Analysis of Cell Surface Proteins

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The expression of surface proteins on cultured cells was measured with flow cytometry. Tumor cells were harvested upon incubation with Accutase (Nacalai Tesque). Cells were stained using CD133/1 (AC133) conjugated to allophycocyanin (APC; 130-090-826; Miltenyi Biotec, Auburn, CA) and CD44 conjugated to APC/Fire750 (33817; BioLegend, San Diego, CA). Relative fluorescent intensities were measured using an SH800 cell sorter (SONY, Tokyo, Japan). Single cells were sorted using an SH800 cell sorter (SONY). Data were analyzed with FlowJo 10.2 software (FlowJo LLC, Ashland, OR, USA).

Fujino S, & Miyoshi N. (2019). Oct4 Gene Expression in Primary Colorectal Cancer Promotes Liver Metastasis. Stem Cells International, 2019, 7896524.

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