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Fitc rat igg2a

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FITC Rat IgG2a is a fluorescently labeled antibody specific to the rat IgG2a isotype. It can be used for flow cytometry, immunohistochemistry, and other immunoassays.

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Lab products found in correlation

Apc rat igg2a, by BioLegend (4 mentions) Efluor 780, by Thermo Fisher Scientific (3 mentions) Anti cd16 cd32 antibody, by BioLegend (3 mentions) Attune nxt flow cytometer, by Thermo Fisher Scientific (3 mentions) Fluorescein isothiocyanate (fitc), by BioLegend (3 mentions) Pe cyanine7 rat igg2b, by BioLegend (3 mentions) Pe rat igg2b, by BioLegend (3 mentions) Pe cyanine7, by BioLegend (3 mentions) Pe rat igg2a, by BioLegend (2 mentions) Apc rat igg2b, by BioLegend (2 mentions) Facsaria 3 flow cytometer, by BD (1 mentions) Fitc anti mouse cd8a, by BioLegend (1 mentions) Anti mouse cd16 32 antibody, by BioLegend (1 mentions) Zombie nir fixable viability dye, by BioLegend (1 mentions) M csf, by Thermo Fisher Scientific (1 mentions) κ isotype ctrl antibody, by BioLegend (1 mentions) Pe cyanine7 anti mouse f4 80 antibody, by BioLegend (1 mentions) Fitc anti mouse cd206 mmr antibody, by BioLegend (1 mentions) Anti mouse f4 80 fitc, by Thermo Fisher Scientific (1 mentions) Fitc anti mouse f4 80, by BioLegend (1 mentions)

Close spelling variants of this product

Rat IgG2a (27 citations) IgG2a (24 citations) FITC Rat IgG2a κ isotype control (2 citations)

10 protocols using fitc rat igg2a

1

Multicolor Flow Cytometric Analysis

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Cells were stained with Zombie NIR Fixable Viability dye (catalog no. 423106; BioLegend, San Diego, CA, United States), which is non-permeant to live cells but is permeant to cells with compromised membranes, i.e., dead cells. The cells were then pre-incubated with purified anti-mouse CD16/32 antibody (catalog no. 101310; BioLegend) to block non-specific immunoglobulin binding to the Fc receptors. Finally, the cells were incubated with specific antibodies or isotype controls according to the manufacturers’ guidelines. The antibodies used were: Pacific Blue anti-mouse CD45; allophycocyanin-conjugated (APC) anti-mouse CD3; fluorescein isothiocyanate–conjugated (FITC) anti-mouse CD8a; peridinin chlorophyll protein complex–conjugated (PerCP) anti-mouse CD4; FITC anti-mouse CD19; phycoerythrin-conjugated (PE) anti-mouse CD366; Pacific Blue Rat immunoglobulin (Ig)G2b, κ Isotype Ctrl; APC Rat IgG2b, κ Isotype Ctrl; FITC Rat IgG2a, κ Isotype Ctrl; PerCP Rat IgG2b, κ Isotype Ctrl; and PE Rat IgG2a, κ Isotype Ctrl (all from BioLegend). The cells were detected and analyzed using a FACSAria III flow cytometer (BD Biosciences, San Jose, CA, United States); positive cells were defined using an isotype control or a fluorescence minus one control.
Zhang Y., Jiang N., Zhang T., Wang D., Feng Y., Sang X., Yang N, & Chen Q. (2018). Toxoplasma gondii Genotype Determines Tim-3 Expression Levels in Splenic and Circulatory T Cells in Mice. Frontiers in Microbiology, 9, 2967.
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2

Isolation and Culture of Primary Mouse Macrophages

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Primary mouse embryonic fibroblasts (MEFs) were generated and cultured as previously described 16 (link).
BM-derived macrophages were generated as previously described 17 (link), 18 (link). Briefly, lineage-committed cells were depleted using FITC-anti-CD3 (IC: FITC-Rat IgG2b, κ, 17A2, BioLegend), FITC-anti-CD19 (IC: FITC-Rat IgG2a, κ, 6D5, BioLegend), FITC-anti-Ly6G (FITC-Rat IgG2a, κ, 1A8, BioLegend) and FITC-anti-TER119 (, IC: FITC-IgG2b, κ, TER-119, eBioscicence) antibodies. Lineage- whole BM cells were cultured in DMEM supplemented with 10% FBS and 20 ng/mL murine recombinant macrophage-colony stimulating factor (M-CSF, Peprotech) for 4 days.
Li R., Li X., Zhao J., Meng F., Yao C., Bao E., Sun N., Chen X., Cheng W., Hua H., Li X., Wang B., Wang H., Pan X., You H., Yang J, & Ikezoe T. (2022). Mitochondrial STAT3 exacerbates LPS-induced sepsis by driving CPT1a-mediated fatty acid oxidation. Theranostics, 12(2), 976-998.
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3

Skin Wound Immune Cell Analysis

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Single cells digested from skin wounds were pre-incubated with purified anti-CD16/CD32 antibody (101301, BioLegend) (1.0 μg per 106 cells in 100 μl volume) for 5 to 10 min to block Fc receptors. The cell suspensions were then co-incubated with fixable viability dye (eFluor™ 780, 65-0865-14, eBioscience) and antibodies against surface markers CD45 (PE/Cyanine7, 147703, BioLegend), CD3 (PE, 100205, BioLegend), and F4/80 (FITC, 123107, BioLegend) at 1:400 dilution for 30 min at 4 °C in the dark (100 μl per antibody per sample). After fixation and permeabilization, cells were incubated with antibodies against intracellular marker CD68 (APC, 137007, Biolegend) at 1:400 dilution for 20 min at 4 °C in the dark (100 μl per antibody per sample). Isotype controls of CD45 (PE/Cyanine7 Rat IgG2b, κ, 400617, Biolegend), CD3 (PE Rat IgG2b, κ, 400607, Biolegend), F4/80 (FITC Rat IgG2a, κ, 400505, BioLegend) and CD68 (APC Rat IgG2a, κ, 400511, Biolegend) were used in same concentration. Flow cytometry analysis was performed using Attune Nxt flow cytometer (Thermo Fisher Scientific) and FlowJo (v10.8.1). The experiments were performed three times independently (n = 3).
Yang Y., Chu C., Liu L., Wang C., Hu C., Rung S., Man Y, & Qu Y. (2023). Tracing immune cells around biomaterials with spatial anchors during large-scale wound regeneration. Nature Communications, 14, 5995.
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4

Macrophage Immunophenotyping by Flow Cytometry

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Primary BMDMs were induced under specific conditions and labeled with the following antibodies for identification of M1 or M2 macrophages: PE/Cyanine7 anti-mouse F4/80 Antibody (Biolegend, 123113) PE/Cyanine5 anti-mouse CD86 Antibody (Biolegend, 105015), FITC anti-mouse CD206 (MMR) Antibody (Biolegend, 141703). Related isotypes for control are PE/Cyanine7 Rat IgG2a, κ Isotype Ctrl Antibody (Biolegend, 400521), PE/Cyanine5 Rat IgG2a, κ Isotype Ctrl Antibody (Biolegend, 400509), FITC Rat IgG2a, κ Isotype Ctrl Antibody (Biolegend, 400505). Cells were suspended and incubated with antibodies for 30 min, and analyzed via Guava Easycyte 12HT (Luminex).
Liu Y., Hao R., Lv J., Yuan J., Wang X., Xu C., Ma D., Duan Z., Zhang B., Dai L., Cheng Y., Lu W, & Zhang X. (2024). Targeted knockdown of PGAM5 in synovial macrophages efficiently alleviates osteoarthritis. Bone Research, 12, 15.
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5

Macrophage Polarization Analysis by Flow Cytometry

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RAW264.7 cells in the logarithmic growth phase were spread to a 6-well plate at 106 cells/well. After at least 6 h, the cells were completely attached to the wall, and the lysate and control were added separately. After 24 h of treatment, RAW264.7 cells were stained with FITC anti-mouse F4/80 (clone: FJK-16s, Invitrogen, Waltham, Massachusetts), APC anti-mouse CD86 (clone: GL-1, Biolegend, San Diego, CA) and PE anti-mouse CD206 (clone: MR6F3, Invitrogen, Waltham, Massachusetts) according to the protocol of the antibodies (M1 macrophage: F4/80+CD86+, M2 macrophage: F4/80+CD206+) and subjected to flow cytometry (LSR II, BD). Detection of SIRPα on macrophages in spleen from mouse used FITC anti-mouse F4/80, APC anti-mouse CD11b (clone: M1/70, Biolegend, San Diego, CA), and PE anti-mouse SIRPα (clone: P84, Biolegend, San Diego, CA). All flow cytometry used to detect macrophage typing in this study was set up with an antibody isotype control group (PE Rat IgG2a, clone: RTK2758, Biolegend, USA; APC Rat IgG2a, clone: RTK2758, Biolegend, USA; FITC Rat IgG2a, clone: RTK2758, Biolegend, USA).
Kong D., Yang Z., Li G., Wu Q., Gu Z., Wan D., Zhang Q., Zhang X., Cheng S., Liu B., Zhang K, & Zhang W. (2022). SIRPα antibody combined with oncolytic virus OH2 protects against tumours by activating innate immunity and reprogramming the tumour immune microenvironment. BMC Medicine, 20, 376.
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6

Cell Surface Integrin Expression Analysis

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Cells were labeled in PAB buffer (phosphate-buffered saline [PBS] + 1% BSA + 0.02% sodium azide) for 30 min on ice with either Alexa Fluor 647 CD82 (clone ASL-24; BioLegend, San Diego, CA), Alexa Flour 488 integrin α4 (clone 7.2R; R&D, Minneapolis, MN), FITC integrin α6 (clone GoH3; BioLegend), Alexa Flour 488 integrin α3 (clone ASC-1; BioLegend), APC integrin α5 (clone NKI-SAM-1; BioLegend), PE integrin α2 (clone HAS3; R&D), FITC integrin β7 (clone FIB27; BioLegend), or Alexa Flour 647 integrin β1 (clone TS2/16; BioLegend). Separate tubes of cells were labeled with Alexa Flour 488 mouse immunoglobulin G1 (IgG1), κ, isotype control (clone 11711; R&D), FITC rat IgG2a, κ, isotype control (clone RTK2758; BioLegend), Alexa Flour 647 mouse IgG1, κ, isotype control (clone MOPC-21; BioLegend), PE mouse IgG2a, κ, isotype control (clone MOPC-173; BioLegend), or APC mouse IgG2b, κ, isotype control (clone MPC-11; BioLegend). Cells were washed three times with PAB buffer and analyzed using Accuri C6 flow cytometer. Histograms were created using FlowJo software; fluorescence values were normalized to the mode.
Termini C.M., Cotter M.L., Marjon K.D., Buranda T., Lidke K.A, & Gillette J.M. (2014). The membrane scaffold CD82 regulates cell adhesion by altering α4 integrin stability and molecular density. Molecular Biology of the Cell, 25(10), 1560-1573.
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7

Monoclonal Antibodies for Immune Cell Analyses

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MAb to C3a receptor (C3aR) (cat# sc-133172, clone D-12, IgG2a, κ, 1:50) was purchased from Santa Cruz Biotechnology, Dallas, TX. MAbs against mouse CD45 (cat# 103128, clone 30-F11, rat IgG2b, κ, 1:100), C5a receptor-1 (C5aR1, CD88) (cat# 135808, clone 20/70, rat IgG2b, κ, 1:100), IL-17A (cat# 506908, clone TC11–18H10.1, rat IgG1, κ, 1:100), CD4 (cat# 100412, clone GK1.5, rat IgG2b, κ, 1:100), TCRβ (cat# 109224, clone H57–597, armenian hamster IgG, 1:100), IL-6Rα chain (CD126) (cat# 115815, clone D7715A7, rat IgG2b, κ, 5 μg/mouse), Ly6G (cat# 127616, clone 1A8, rat IgG2a, κ, 1:100), EpCAM (cat# 118208, clone G8.8, rat IgG2a, κ, 1:100), rat IgG2b, κ isotype control (cat# 400644, clone RTK4530, 1:100 or 5 μg/mouse), PE mouse IgG2a, κ isotype control (cat# 400213, clone MOPC-173, 1:50), APC rat IgG2b, κ isotype control (cat# 400611, clone RTK4530, 1:100), Alexa Fluor 700 rat IgG2b, κ isotype control (cat# 400628, clone RTK4530, 1:100), FITC rat IgG2a, κ isotype control (cat# 400505, clone RTK2758, 1:100), were from Biolegend, San Diego, CA. MAbs to EpCAM (cat# 563478, clone G8.8, rat IgG2a, κ, 1:100), IL-6 (cat# 554401, clone MP5–20F3, rat IgG1, 1:100), APC rat IgG2a κ isotype control (cat# 554690, clone R35–95, 1:100) and PE rat IgG1, κ isotype control (cat# 554685, clone R3–34, 1:100) were obtained from BD Bioscience, Franklin Lakes, NJ.
Wang H., Ideguchi H., Kajikawa T., Mastellos D.C., Lambris J.D, & Hajishengallis G. (2022). Complement is required for microbe-driven induction of Th17 and periodontitis. Journal of immunology (Baltimore, Md. : 1950), 209(7), 1370-1378.
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8

Skin Wound Immune Cell Analysis

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Single cells digested from skin wounds were pre-incubated with purified anti-CD16/CD32 antibody (101301, BioLegend) (1.0 μg per 106 cells in 100 μl volume) for 5 to 10 min to block Fc receptors. The cell suspensions were then co-incubated with fixable viability dye (eFluor™ 780, 65-0865-14, eBioscience) and antibodies against surface markers CD45 (PE/Cyanine7, 147703, BioLegend), CD3 (PE, 100205, BioLegend), and F4/80 (FITC, 123107, BioLegend) at 1:400 dilution for 30 min at 4 °C in the dark (100 μl per antibody per sample). After fixation and permeabilization, cells were incubated with antibodies against intracellular marker CD68 (APC, 137007, Biolegend) at 1:400 dilution for 20 min at 4 °C in the dark (100 μl per antibody per sample). Isotype controls of CD45 (PE/Cyanine7 Rat IgG2b, κ, 400617, Biolegend), CD3 (PE Rat IgG2b, κ, 400607, Biolegend), F4/80 (FITC Rat IgG2a, κ, 400505, BioLegend) and CD68 (APC Rat IgG2a, κ, 400511, Biolegend) were used in same concentration. Flow cytometry analysis was performed using Attune Nxt flow cytometer (Thermo Fisher Scientific) and FlowJo (v10.8.1). The experiments were performed three times independently (n = 3).
Yang Y., Chu C., Liu L., Wang C., Hu C., Rung S., Man Y, & Qu Y. (2023). Tracing immune cells around biomaterials with spatial anchors during large-scale wound regeneration. Nature Communications, 14, 5995.
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9

Skin Wound Immune Cell Analysis

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Single cells digested from skin wounds were pre-incubated with purified anti-CD16/CD32 antibody (101301, BioLegend) (1.0 μg per 106 cells in 100 μl volume) for 5 to 10 min to block Fc receptors. The cell suspensions were then co-incubated with fixable viability dye (eFluor™ 780, 65-0865-14, eBioscience) and antibodies against surface markers CD45 (PE/Cyanine7, 147703, BioLegend), CD3 (PE, 100205, BioLegend), and F4/80 (FITC, 123107, BioLegend) at 1:400 dilution for 30 min at 4 °C in the dark (100 μl per antibody per sample). After fixation and permeabilization, cells were incubated with antibodies against intracellular marker CD68 (APC, 137007, Biolegend) at 1:400 dilution for 20 min at 4 °C in the dark (100 μl per antibody per sample). Isotype controls of CD45 (PE/Cyanine7 Rat IgG2b, κ, 400617, Biolegend), CD3 (PE Rat IgG2b, κ, 400607, Biolegend), F4/80 (FITC Rat IgG2a, κ, 400505, BioLegend) and CD68 (APC Rat IgG2a, κ, 400511, Biolegend) were used in same concentration. Flow cytometry analysis was performed using Attune Nxt flow cytometer (Thermo Fisher Scientific) and FlowJo (v10.8.1). The experiments were performed three times independently (n = 3).
Yang Y., Chu C., Liu L., Wang C., Hu C., Rung S., Man Y, & Qu Y. (2023). Tracing immune cells around biomaterials with spatial anchors during large-scale wound regeneration. Nature Communications, 14, 5995.
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10

Quantifying Kupffer Cell Subsets

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Reagents and antibodies used for flow cytometry were CD45-BV650 clone 30-F11, F4/80-PECy7 clone BM8, CD11b-PE clone M1/70, CD206-FITC C068C2, FITC Rat IgG2a, κ Isotype Ctrl—Clone RTK2758 (Biolegend, San Diego, CA, USA), DAF-FM diacetate (ThermoFisher Scientific, Burlington, ON, Canada), Fixable Viability Dye eFluor® 450 (eBioscience/ThermoFisher Scientific, Burlington, ON, Canada).
Livers were digested and live cells were gated on. The pro-inflammatory NO+ KCs population was evaluated by the following combination: CD45+, CD11blo, F4/80hi, DAF-FM + (the marker for nitric oxide production). The anti-inflammatory CD206+ KCs population was assessed by the following markers: CD45+, CD11blo, F4/80hi, CD206 + (mannose receptor). Additionally, the anti-inflammatory IL-10+ KCs population was assessed by flow cytometry quantifying GFP expression in KCs of Vert-X mouse (CD45+, CD11blo, F4/80hi, GFP-IL10+). The pro-inflammatory-NO+/ anti-inflammatory-CD206+ Kupffer cells ratio was calculated by dividing the percentage of cells positive for NO by the total of cells positive for CD206.
Linares I., Farrokhi K., Echeverri J., Kaths J.M., Kollmann D., Hamar M., Urbanellis P., Ganesh S., Adeyi O.A., Yip P., Selzner M, & Selzner N. (2018). PPAR-gamma activation is associated with reduced liver ischemia-reperfusion injury and altered tissue-resident macrophages polarization in a mouse model. PLoS ONE, 13(4), e0195212.
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