Fitc lineage

Manufactured by BioLegend

Sourced in United States

FITC-lineage is a fluorescent label used in flow cytometry applications. It is designed to conjugate with specific antibodies or other biomolecules, allowing for the detection and analysis of target cells or molecules within a sample.

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Lineage cocktail-FITC (4 citations) FITC anti-mouse Lineage Cocktail (2 citations) FITC anti-Lineage (2 citations)

6 protocols using fitc lineage

Isolation and Analysis of Tumor-Infiltrating Cells

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Tumor tissues were minced and incubated in digestion solution containing 0.5 mg/ml collagenase V, 0.2 mg/ml hyaluronidase and 0.015 mg/ml DNase I (Sigma, St. Louis, USA) in a 37 °C water bath for 1 h.
The samples were then ltered through a 70 µm strainer before staining with uorescence antibodies. Mouse Percy5-CD45, FITC-lineage, PE-CD90.2, and APC-KLRG1 (Biolegend) were used to analyze the percentage of ILC2s in the mouse tumor tissue and human Percy5-CD45, FITC-lineage, PE-CRTH2, and APC-CD127 (Biolegend) were used to analyze the percentage of ILC2s in the human tumor tissue. Mouse CD8 cells were stained with Percy5-CD45, FITC-CD3, and PE-CD8 (Biolegend). For analyzing the percentage of MDSCs, FITC-CD11b and APC-Gr-1 (Biolegend) were used. All surface stained samples were kept at 4 °C for 30 min.
For intracellular staining of IL-9, tumor tissue single-cell suspensions were rst stimulated with 50 ng/mL phorbol myristate acetate (PMA), 1 µg/mL ionomycin, and 2 µg/mL monensin (eBioscience, San Diego, USA) for 6 h. Samples were xed, permeabilized, and stained with IL-9 antibody on a shaker at 4 °C for 45 min.
The apoptosis kit FITC-Annexin V and APC-7-AAD (Multisciences, Hangzhou, China) was used following the manufacturer's protocols to analyze the apoptosis of CT26 cells.

Wan J., Wu Y., Huang L., Tian Y., Ji X., Abdelaziz M.H., Cai W., Dineshkumar K., Lei Y., Yao S., Sun C., Su Z., Wang S, & Xu H. (2021). ILC2-derived IL-9 inhibits colorectal cancer progression by activating CD8⁺ T cells. Cancer letters, 502.

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Characterization of Tumor-Infiltrating Immune Cells

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Tumor tissues were minced and incubated in digestion solution containing 0.5 mg/ml collagenase V, 0.2 mg/ml hyaluronidase and 0.015 mg/ml DNase I (Sigma, St. Louis, USA) in a 37 °C water bath for 1 h. The samples were then filtered through a 70 µm strainer before staining with fluorescence antibodies. Mouse Percy5-CD45, FITC-lineage, PE-CD90.2, and APC-KLRG1 (Biolegend) were used to analyze the percentage of ILC2s in the mouse tumor tissue and human Percy5-CD45, FITC-lineage, PE-CRTH2, and APC-CD127 (Biolegend) were used to analyze the percentage of ILC2s in the human tumor tissue. Mouse CD8 cells were stained with Percy5-CD45, FITC-CD3, and PE-CD8 (Biolegend). For analyzing the percentage of MDSCs, FITC-CD11b and APC-Gr-1 (Biolegend) were used. All surface stained samples were kept at 4 °C for 30 min.
For intracellular staining of IL-9, tumor tissue single-cell suspensions were first stimulated with 50 ng/mL phorbol myristate acetate (PMA), 1 µg/mL ionomycin, and 2 µg/mL monensin (eBioscience, San Diego, USA) for 6 h. Samples were fixed, permeabilized, and stained with IL-9 antibody on a shaker at 4 °C for 45 min.

Wan J., Huang L., Wu Y., Ji X., Yao S., Abdelaziz M.H., Cai W., Wang H., Cheng J., Kesavan D.K., Vasudevan A., Sun C., Su Z., Wang S., & xu h. (2020). ILC2-derived IL-9 inhibit s colorectal cancer progression by activating CD8+ T cells.

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Multicolor Flow Cytometry Panel for Murine HSCs

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Cells were Fc blocked in 1:50 TruStain FcX PLUS (BioLegend, catalog no. 156604) in 2% FBS/PBS for 10 min, followed by a stain of at least 30 min of 1:200 CD11b-FITC (Thermo Fisher Scientific, catalog no. 11-0112-82), 1:400 CD117-PE (Thermo Fisher Scientific, catalog no. 12-1171-82), 1:200 Sca-1-PE/Cy7 (BioLegend, catalog no. 122514), 1:1000 Ghost Dye Red 780 fixable viability dye (Cell Signaling Technology, catalog no. 18452), and/or 1:200 lineage-FITC (BioLegend catalog no. 133301). Cells were then washed twice with 2% FBS/PBS and run on the flow cytometer. For compensation, UltraComp eBeads compensation beads (Thermo Fisher Scientific, catalog no. 01-2222-42) were stained at the same concentration for the same amount of time as the cells to be analyzed, washed twice with 2% FBS/PBS, and run on the flow cytometer. For viability dye compensation, an aliquot of cells was stained only with 1:1000 Ghost Dye Red 780 fixable viability dye (Cell Signaling Technology, catalog no. 18452) for the same duration as the cells to be analyzed, washed twice with 2% FBS/PBS, and run on the flow cytometer.

Greenwood D.L., Ramsey H.E., Nguyen P.T., Patterson A.R., Voss K., Bader J.E., Sugiura A., Bacigalupa Z.A., Schaefer S., Ye X., Dahunsi D.O., Madden M.Z., Wellen K.E., Savona M.R., Ferrell P.B, & Rathmell J.C. (2022). Acly Deficiency Enhances Myelopoiesis through Acetyl Coenzyme A and Metabolic–Epigenetic Cross-Talk. ImmunoHorizons, 6(12), 837-850.

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Phenotyping Lung and Colon ILC2 Cells

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Flow cytometry was used to detect the ILC2 cells in the lung and colon tissues. Briefly, the mouse lung/colon single-cell suspension in PBS containing 1% BSA was obtained from lung/colon tissues after homogenization, digestion and centrifugation. The cell suspension (100 µL; cell concentration 1 × 10⁶ cells/mL) was incubated with the following antibodies (all from BD unless mentioned): the APC-CY7-CD45 (2D1), Lineage-FITC, CD3 (UCHT1), CD19 (HIB19), CD123 (7G3), CD11b (M1/70), CD11c (B-ly6), CD8 (RPA-T8), FceRI (AER-37 (CRA-1), CD14 (M5E2), CD4 (RPA-T4), CD56 (B159), PerCP-CY5.5-90.2 (30-H12, BioLegend, San Diego, CA, USA), PE-CY7-ST2 (DIH4), and PE-CD127 (SB/199), at room temperature for 30 min in dark. After washing with PBS twice, flow cytometry was performed to detect the ratio of Lin⁻CD45⁺CD127⁺ST2⁺KLRG1^int cells and Lin⁻CD45⁺CD127⁺ST2⁻KLRG1^hi cells with the cytometer (LSR II, BD, USA). The data were analyzed using the Kaluza software (Beckman Coulter, Inc).

Jiang M., Li Z., Zhang F., Li Z., Xu D., Jing J., Li F., Wang J, & Ding J. (2023). Butyrate inhibits iILC2-mediated lung inflammation via lung-gut axis in chronic obstructive pulmonary disease (COPD). BMC Pulmonary Medicine, 23, 163.

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Murine Hematopoietic Stem Cell Isolation

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Cells were harvested from femurs and tibias. Red blood cells were lysed using 0.16 M ammonium chloride. Cells were stained using the following antibodies: FITC lineage, BioLegend (CD3e, clone 145-2C11, CD4, clone GK1.5, CD8a, clone 53-6.7, B220, IgM, clone RMM-1, Mac-1, clone M1/70, Gr1, clone RB6-8C5, Ter119 respectively); PE-Cy7 Sca-1 (clone D7, BioLegend); APC c-Kit (clone ACK2, BioLegend; BV711 CD150 (clone TC15-12F12.2, BioLegend); PerCP Cy5.5 CD48 (clone HM48-1, BioLegend); BV421 human CD45 (clone HI30, BioLegend) [58 (link)]. Flow cytometry analysis was performed using a custom LSR Fortessa X-20 analyzer (BD Biosciences). Data analysis was performed using FlowJo software (BD Biosciences). Mouse HSCs were defined as lineage negative, Sca-1+, c-Kit+, CD150+, CD48− cells. Cellularity was based on the total nucleated cell count from 1 femur + 1 tibia.

Chugh R.M., Bhanja P., Olea X.D., Tao F., Schroeder K., Zitter R., Arora T., Pathak H., Kimler B.F., Godwin A.K., Perry J.M, & Saha S. (2022). Human Peripheral Blood Mononucleocyte Derived Myeloid Committed Progenitor Cells Mitigate H-ARS by Exosomal Paracrine Signal. International Journal of Molecular Sciences, 23(10), 5498.

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Flow Cytometry Analysis of Immune Cells

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Spleen, mesenteric lymph node (mLN), and ankle joints were processed for flow cytometry as we previously described [13 (link)]. The following antibodies were used for surface molecular staining: BV510-anti-CD45 (30F11), FITC-CD4 (JK1.5), FITC-Lineage (CD3/GR-1/CD11b/CD45R/TER-119), BV421-CD127 (A7R34), PerCP/Cy5.5-KLRG1 (2F1), PE-ST2 (RMST2-2), APC-ICOS (C398.4A), APC-NK1.1 (PK136), PerCP/Cy5.5-Ly6G(1A8), FITC-CD11b(M1/70), APC-F4/80 (BM8, all from BioLegend), and PE-SiglecF (1RNM44N, eBioscience). For intracellular staining, cells were fixed and permeabilized by the FoxP3/Transcription Factor Staining Buffer (eBioscience) and then stained with PE/Cy7-anti-T-bet (4B10), PE-anti-RORγt (AFKGS-9), PerCP/Cy5.5-IFN-γ (XMG1.2, BD Biosciences), APC-IL-4 (11B11, BioLegend), and AF647-IL-17A (TC11-18H10, BD Biosciences) at 4°C for 30 min. In some experiments, mLN cells and splenocytes were plated on a 12-well plate and incubated with cell activation cocktail (with Brefeldin A, BioLegend) at 37°C for 4 h before staining.

Zhang Y., Qin Y, & Chen Z. (2021). Neuromedin U Suppresses Collagen-Induced Arthritis through ILC2-Th2 Activation. Journal of Immunology Research, 2021, 5599439.

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