Pe anti mouse ifn γ clone xmg1, by BioLegend - Product details

1

Multiparameter Flow Cytometry Analysis

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Single cell suspensions were pretreated with mouse Fc-Block™ (BD Biosciences/ Pharmingen) and incubated with fluorophore-labeled antibodies or appropriate isotype controls. Acquisitions were performed using a BD FACSCanto™ II (BD Bioscience). For intracellular staining of cytokines BD Cytofix/Cytoperm (BD Biosciences) was used. Analysis was done using BD FACSDiva™ and FlowJo (Treestar INC.) software. Following antibodies were used: Phycoerythrin-(PE), allophycocyanin-(APC) and PE/Cy7 anti-human FcεRIα mAb CRA1 (clone AER-37, eBioscience or BioLegend), PE-anti-mouse IgE (RME-1, Biolegend), APC-anti-CD11c, Alexa Fluor® 647 anti-mouse CD8α and Alexa Fluor® 647 anti-mouse CD4 from Biolegend (San Diego, CA), PE-anti-mouse CD86 (BD Pharmingen), APC-anti- CD117 (Biolegend), and PE- anti-mouse FcεRIα mAb (Mar1, Biolegend). Intracellular cytokine stain included PE anti-mouse IFN-γ (clone XMG1.2, Biolegend) and APC anti-mouse IL-4 (clone 11B11, eBioscience). Anti-human CD1c (BDCA-1, clone AD5-8E7, Miltenyi Biotech), APC-anti-human CD19 (clone HIB19, BioLegend), PE/Cy7 anti- human IgE (clone MHE-18, BioLegend), PE anti-human CD203 (Biolegend), Alexa Fluor® anti human CD83 (clone HB15e), PE anti-human CD80 (Biolegend) and APC anti-human CD1a (BD Pharmingen).

Platzer B., Baker K., Vera M.P., Singer K., Panduro M., Lexmond W.S., Turner D., Vargas S.O., Kinet J.P., Maurer D., Baron R.M., Blumberg R.S, & Fiebiger E. (2014). Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites. Mucosal immunology, 8(3), 516-532.

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2

Cytokine Profiling of CD8+ T Cells

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Single cell suspensions were prepared from spleens. 1 x 10⁶ cells were cocultured in round bottom 96 well plates with 1 μg/ml H-2Ld IE peptide YPHFMPTNL or, as negative control, scrambled peptide NFYPTLPHM for 1 h. After that, Brefeldin A (Biolegend) was added at a concentration of 5 μg/ml and the coculture continued for 5 h. Cells were stained for CD8 (FITC anti-mouse CD8a, clone 53–6.7; Biolegend), fixed in 1% paraformaldehyde, permeabilized with 0.1% saponin (Sigma-Aldrich), stained for intracytoplasmic IFN-γ (PE anti-mouse IFN-γ, clone XMG1.2; Biolegend), analyzed on a FACSCalibur and depicted using FlowJo software (BD Biosciences).

Eletreby M., Thiessen L., Prager A., Brizic I., Materljan J., Kubic L., Jäger K., Jurinović K., Jerak J., Krey K, & Adler B. (2023). Dissecting the cytomegalovirus CC chemokine: Chemokine activity and gHgLchemokine-dependent cell tropism are independent players in CMV infection. PLOS Pathogens, 19(12), e1011793.

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3

OT-I Memory CD8+ T Cell Differentiation

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Splenocytes from OT-I mice were obtained aseptically. Cells were cultured in 96-well plate (10⁵ cells/well) and activated with SIINFEKL peptide (4 × 10⁻⁹ M) in the presence or absence of SCFAs (400 μM) for 3 days. Then cells were washed 3 times with T cell medium and further cultured with interleukin-15 (IL-15, 10 ng/mL, from PeproTech, Rocky Hill, NJ) in the presence or absence of SCFAs for 3 days to generate of OVA-specific OT-I memory CD8+ T cells. Thereafter, cells were washed twice and re-activated with SIINFEKL peptide (10 μM) for 2 h. Subsequently, brefeldin A was added (1 : 1000 dilution) and incubated for another 2 h. The suspension cells were collected, blocked with CD16/32 antibody, and stained with fixable viability dye eFluor™ 450, FITC-CD44 Rat anti-Human/Mouse, PE-Cy7-CD62L Monoclonal Antibody. Intracellular IFN-γ (PE-anti-mouse IFN-γ, clone: XMG1.2, Biolegend) and Tcf1 (PE-Tcf1/Tcf7 Rabbit mAb) positive T cells were detected via flow cytometry.

Han K., Nam J., Xu J., Sun X., Huang X., Animasahun O., Achreja A., Jeon J.H., Pursley B., Kamada N., Chen G.Y., Nagrath D, & Moon J.J. (2021). Generation of systemic antitumour immunity via the in situ modulation of the gut microbiome by an orally administered inulin gel. Nature biomedical engineering, 5(11), 1377-1388.

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4

Multiparameter Flow Cytometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Single cell suspensions were pretreated with mouse Fc-Block™ (BD Biosciences/ Pharmingen) and incubated with fluorophore-labeled antibodies or appropriate isotype controls. Acquisitions were performed using a BD FACSCanto™ II (BD Bioscience). For intracellular staining of cytokines BD Cytofix/Cytoperm (BD Biosciences) was used. Analysis was done using BD FACSDiva™ and FlowJo (Treestar INC.) software. Following antibodies were used: Phycoerythrin-(PE), allophycocyanin-(APC) and PE/Cy7 anti-human FcεRIα mAb CRA1 (clone AER-37, eBioscience or BioLegend), PE-anti-mouse IgE (RME-1, Biolegend), APC-anti-CD11c, Alexa Fluor® 647 anti-mouse CD8α and Alexa Fluor® 647 anti-mouse CD4 from Biolegend (San Diego, CA), PE-anti-mouse CD86 (BD Pharmingen), APC-anti- CD117 (Biolegend), and PE- anti-mouse FcεRIα mAb (Mar1, Biolegend). Intracellular cytokine stain included PE anti-mouse IFN-γ (clone XMG1.2, Biolegend) and APC anti-mouse IL-4 (clone 11B11, eBioscience). Anti-human CD1c (BDCA-1, clone AD5-8E7, Miltenyi Biotech), APC-anti-human CD19 (clone HIB19, BioLegend), PE/Cy7 anti- human IgE (clone MHE-18, BioLegend), PE anti-human CD203 (Biolegend), Alexa Fluor® anti human CD83 (clone HB15e), PE anti-human CD80 (Biolegend) and APC anti-human CD1a (BD Pharmingen).

Platzer B., Baker K., Vera M.P., Singer K., Panduro M., Lexmond W.S., Turner D., Vargas S.O., Kinet J.P., Maurer D., Baron R.M., Blumberg R.S, & Fiebiger E. (2014). Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites. Mucosal immunology, 8(3), 516-532.

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5

OT-I Memory CD8+ T Cell Differentiation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Splenocytes from OT-I mice were obtained aseptically. Cells were cultured in 96-well plate (10⁵ cells/well) and activated with SIINFEKL peptide (4 × 10⁻⁹ M) in the presence or absence of SCFAs (400 μM) for 3 days. Then cells were washed 3 times with T cell medium and further cultured with interleukin-15 (IL-15, 10 ng/mL, from PeproTech, Rocky Hill, NJ) in the presence or absence of SCFAs for 3 days to generate of OVA-specific OT-I memory CD8+ T cells. Thereafter, cells were washed twice and re-activated with SIINFEKL peptide (10 μM) for 2 h. Subsequently, brefeldin A was added (1 : 1000 dilution) and incubated for another 2 h. The suspension cells were collected, blocked with CD16/32 antibody, and stained with fixable viability dye eFluor™ 450, FITC-CD44 Rat anti-Human/Mouse, PE-Cy7-CD62L Monoclonal Antibody. Intracellular IFN-γ (PE-anti-mouse IFN-γ, clone: XMG1.2, Biolegend) and Tcf1 (PE-Tcf1/Tcf7 Rabbit mAb) positive T cells were detected via flow cytometry.

Han K., Nam J., Xu J., Sun X., Huang X., Animasahun O., Achreja A., Jeon J.H., Pursley B., Kamada N., Chen G.Y., Nagrath D, & Moon J.J. (2021). Generation of systemic antitumour immunity via the in situ modulation of the gut microbiome by an orally administered inulin gel. Nature biomedical engineering, 5(11), 1377-1388.

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Pe anti mouse ifn γ clone xmg1

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5 protocols using pe anti mouse ifn γ clone xmg1

Multiparameter Flow Cytometry Analysis

Cytokine Profiling of CD8+ T Cells

OT-I Memory CD8+ T Cell Differentiation

Multiparameter Flow Cytometry Analysis

OT-I Memory CD8+ T Cell Differentiation

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