Apc mouse igg1 κ isotype control

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The APC mouse IgG1 κ isotype control is a laboratory reagent used as a reference or background control in flow cytometry experiments. It is a purified mouse immunoglobulin G1 (IgG1) kappa light chain antibody conjugated to the fluorochrome allophycocyanin (APC). This isotype control helps to distinguish specific antibody binding from non-specific binding in flow cytometry applications.

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7 protocols using apc mouse igg1 κ isotype control

Comprehensive Immunophenotyping of Epithelial Cells

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1 × 10⁵ epithelial target cells were washed and re-suspended in the cell staining buffer (Biolegend) containing different dye-conjugated primary antibodies at 1 μg/ml or 5 μg/ml for 30 min at 4°C. Cells were then washed and stained with the Zombie NIR (cell death marker, Biolegend). The stained cells were analyzed by flow cytometry with a FACSCanto II cytometer (BD Biosciences). Primary antibodies targeting the various surface ligands and receptors as well as the corresponding control include: FITC anti-human HLA-A,B,C (Clone W6/32, Biolegend), PE anti-human HLA-E (Clone 3D12, Biolegend), FITC anti-human ICAM-1/CD54 (Clone DX2, Biolegend), FITC anti-human Fas/CD95 (Clone DX2, Biolegend), APC anti-human Nectin-2/CD112 (Clone TX31, Biolegend), APC anti-human PVR/CD155 (Clone SKII.4, Biolegend), PE anti-human MICA (Clone 159227, R&D Systems), PE anti-human MICB (Clone 236511, R&D Systems), PE anti-human ULBP-2/5/6 (Clone 165903, R&D Systems), FITC Mouse IgG1 κ Isotype Control (Clone MOPC-21, Biolegend), APC Mouse IgG1 κ Isotype Control (Clone MOPC-21, Biolegend), and PE Mouse IgG2b κ Isotype Control (Clone MPC-11, Biolegend). Cell death analysis by Annexin V staining was performed using the FITC Annexin V detection kit (Biolegned) following the manufacturer’s protocol.

Zhu Y., Xie J, & Shi J. (2021). Rac1/ROCK-driven membrane dynamics promote natural killer cell cytotoxicity via granzyme-induced necroptosis. BMC Biology, 19, 140.

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Activation Markers on Memory CD4+ T Cells

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The activation markers expression on resting memory CD4⁺ T cells were detected after IP-10 or CD3/CD28 stimulated and measured using the following fluorophore-conjugated antibodies: phycoerythrin (PE)-Cy7 anti-human CD3, allophycocyanin (APC)-Cy7 anti-human CD4, peridinin-chlorophyll-protein anti-human CD197/CCR7, fluorescein isothiocyanate (FITC) anti-human CD45RA, APC anti-human CD69, APC anti-human CD25, APC anti-human HLA-DR, APC mouse IgG1 κ isotype control, and PE mouse IgG1 κ isotype control (all from Biolegend). Samples were analyzed using an LSR II flow cytometer (BD Biosciences), which was adjusted with 10-peak color rainbow beads (Spherotech, Lake Forest, IL, USA). Gates were defined using appropriate isotype controls. Expression levels in each sample were analyzed with FlowJo v10 software (Ashland, OR, USA).

Wang Z., Yin X., Ma M., Ge H., Lang B., Sun H., He S., Fu Y., Sun Y., Yu X., Zhang Z., Cui H., Han X., Xu J., Ding H., Chu Z., Shang H., Wu Y, & Jiang Y. (2021). IP-10 Promotes Latent HIV Infection in Resting Memory CD4+ T Cells via LIMK-Cofilin Pathway. Frontiers in Immunology, 12, 656663.

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Mesenchymal Stem Cell Characterization

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TGF-β1 (PEPRO TECH, China, 100-21-10 μg), PC-MSC (Provided by Shenzhen 150 Biomedical Co.,Ltd.), ESC (Provided by ATCC), FITC Mouse IgG1 κ Isotype control (Biolegend, 7313762), PE Mouse IgG1 κ Isotype control (Biolegend, 9217868), APC Mouse IgG1 κ Isotype control (Biolegend, 9282592), PE Mouse Anti-Human CD73 (Biolegend, 8151901), APC Mouse Anti-Human CD105 (Biolegend, 9277133), FITC Mouse IgG2a κ Isotype control (Biolegend, 8241935), FITC Mouse Anti-Human CD14 (Biolegend, 8299630), FITC Mouse Anti-Human CD45 (Biolegend, 8206704), DMEM/F12 medium (Peiyuan, Shanghai, L310 kJ), Fetal bovine serum (Gemini, 900-108), Trypsin digestive fluid (Beyo, C0203), DPBS (Beyo, C0221G), CCK8 (Beyotime, C0039), EDU staining kit (Beyotime, C0081s), Hematoxylin-eosin staining kit (Solarbio, G1120), DEPC-treated water (Solarbio, R1600), trichloromethane (Sinopharm Chemical Reagent Co. LTD., 10006818), GelRed dye (Biotium, #41003), RNA Loading Buffer (TaKaRa, 9168), Tris-Borate-EDTA Buffer (TBE) 10× Powder, pH8.3 (TaKaRa, T9122), RNAsimple Total RNA Kit (TIANGEN, DP419), All-in-One First-Strand Synthesis Master Mix (Yugong Biolabs, EG15133S), SuperReal PreMix Plus (SYBR Green) (TIANGEN, FP205-02), and Methacrylate anhydride (MAA, 0.1 mL/1 g gelatin), ELISA Kit (Solarbio, SEKH-0052), MateRegen^® Gel (2104006, BioRegen Biomedical (Changzhou) Co., Ltd.).

Zhu Y., Wang T., Bao M.J., Qu X.H, & Li Z.M. (2023). Effect of stem cell conditional medium-loading adhesive hydrogel on TGF-β1-induced endometrial stromal cell fibrosis. Frontiers in Bioengineering and Biotechnology, 11, 1168136.

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Neutrophil Subgroup Characterization

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After different neutrophil subgroups were obtained from step 2.2.1.2, typically 1 × 10⁵ cell/50 μl and the following anti-human fluorochrome-conjugated mAbs or specific isotype controls: CD66b (Biolegend, USA), PE-Cy7 mouse anti-human CD10 (Biolegend, USA), PerCP-vio700 mouse anti-human CD11b (Miltenyi, German), APC mouse anti-human CD16 (Biolegend, USA), PE mouse IgG₁κ isotype control (Biolegend, USA), PE-Cy7 mouse IgG₁κ isotype control (Biolegend, USA), PerCP-vio700 mouse IgG₁ Isotype (Miltenyi, German), and APC mouse IgG₁κ isotype control (Biolegend, USA) 10 μl, respectively were intubated for 15 min in the dark room at room temperature, 500 μl RBC Lysis Buffer (Macs, German) was added into the tube. The mixture was centrifuged with 2,000 rpm for 5 min after another intubating for 15 min in the dark room at room temperature, then removed the supernatant and added 500 μl PBS fluid into the precipitate to resuspend cells which was analyzed by eight-color three-laser-MACSQuant Analyzer (Miltenyi Biotec, German) while data analysis performed using FlowJo software (Tree Star, USA).

Liu M., Wang G., Wang L., Wang Y., Bian Y., Shi H, & Liu J. (2023). Immunoregulatory functions of mature CD10+ and immature CD10– neutrophils in sepsis patients. Frontiers in Medicine, 9, 1100756.

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Isolation and Characterization of Human Platelets

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Platelets were isolated by differential centrifugation as previously reported
¹⁹ (link) with some modifications. Briefly, human blood was collected from healthy volunteers who had not received any medications in the last 2 weeks. The blood was transferred to tubes containing 1/9 volume of anticoagulation citrate‐dextrose solution (Sigma‐Aldrich), and centrifugation at 100 g was carried out for 20 min to obtain PRP. The PRP was centrifuged at 100 g for 15 min to pellet red and white blood cells, and the supernatants were centrifuged at 800 g for another 15 min to obtain platelets and quantified using a hemacytometer. A portion of platelets was incubated with APC antihuman CD61 Ab (B312450; BioLegend), followed by purity analysis using the FACSAria II system (Becton Dickinson). The APC mouse IgG1 κ isotype control (B344277; BioLegend) was used as the isotype control (Figure S1–S2). Another portion of the platelets was stained using PKH67 Green Fluorescent Cell Linker Kits (Sigma‐Aldrich) according to the manufacturer's protocol and used in the biological assays.

Yu L., Hong Y., Maishi N., Matsuda A.Y., Hida Y., Hasebe A., Kitagawa Y, & Hida K. (2023). Oral bacterium Streptococcus mutans promotes tumor metastasis through thrombosis formation. Cancer Science, 115(2), 648-659.

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NK Cell Cytotoxicity Assay with Engineered Raji Cells

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Raji β2-microglobulin knockout and HHLA2 transfected Raji β2-microglobulin knockout cells were co-cultured in RPMI1640 medium supplemented with 10% FBS, 1% Gluta-Max and 1% Penicillin/Streptomycin with NK-92 MI cells for 3 hours at E:T ratios of 1:1, 2:1 and 3:1 in round-bottom 96-well plates at 37°C with 5% CO₂. Anti-KIR3DL3 (1G7) and mouse IgG2b isotype control (Bxcell; cat. #BE0086) were used at 10μg/ml. BV421 anti-human CD107a (Biolegend; cat. #328625), APC anti-human CD56 (Biolegend; cat. #362503), APC Mouse IgG1, κ Isotype control (Biolegend; cat. #400121) and BV421 Mouse IgG1, κ Isotype control (Biolegend; cat. #400157) were used at 1:100 dilution. Monensin Solution (1,000X) (Biolegend; cat 420701) was used at 1:1000 dilution and Cell Stimulation Cocktail, PMA/Ionomycin (Biolgend; cat. #423301) was used at 1:500 dilution. Subsequently the plate was put on ice and cells were analyzed for CD107a expression on CD56 gated cells using flow cytometry and FlowJo software.

Bhatt R.S., Berjis A., Konge J.C., Mahoney K.M., Klee A.N., Freeman S.S., Chen C.H., Jegede O.A., Catalano P.J., Pignon J.C., Sticco-Ivins M., Zhu B., Hua P., Soden J., Zhu J., McDermott D.F., Arulanandam A.R., Signoretti S, & Freeman G.J. (2020). KIR3DL3 is an inhibitory receptor for HHLA2 that mediates an alternative immunoinhibitory pathway to PD1. Cancer immunology research, 9(2), 156-169.

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Phenotypic Analysis of Neutrophils

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Added 100 μl peripheral anticoagulant blood in healthy volunteer or sepsis patient into the tube with following anti-human fluorochrome-conjugated mAbs or specific isotype controls: PE mouse anti-human CD66b (Biolegend, USA), PE-Cy7 mouse anti-human CD10 (Biolegend, USA), PE mouse IgG₁κ isotype control (Biolegend, USA), PE-Cy7 mouse IgG₁κ isotype control (Biolegend, USA), PerCP-vio700 mouse IgG₁ Isotype (Miltenyi, German), and APC mouse IgG₁κ isotype control (Biolegend, USA) 10 μl, respectively. After intubating for 15 min in the dark room at room temperature, 500 μl RBC Lysis Buffer (Macs, German) was added into the tube. The mixture was centrifuged with 2,000 rpm for 5 min after another intubating for 15 min in the dark room at room temperature, then removed the supernatant and added 500 μl PBS fluid into the precipitate to resuspend cells which was analyzed by eight-color three-laser-MACSQuant Analyzer (Miltenyi Biotec, German) while data analysis was performed by using FlowJo software (Tree Star, USA).

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