Mouse splenocytes were collected aseptically from spleens of mice by mincing the spleen tissues in a sterile Petri plate, and the erythrocytes were lysed in lysis buffer (10 mmol/L KHCO3, 150 mmol/L NH4Cl, 10 mmol/L ethylenediaminetetraacetic acid, pH 7.4). Splenocytes were prepared from tumor-grafted mice or naïve mice. ACK (Ammonium–Chloride–Potassium) Lysing buffer was used to lyse erythrocytes. Splenocytes were stained using APC-conjugated anti-mouse CD3e antibody, APC-conjugated anti-mouse CD4, FITC-conjugated anti-mouse CD8a, APC/Cy7-conjugated anti-mouse CD11b, APC-conjugated anti-mouse CD45, and PE-conjugated anti-mouse Ly6G/Ly6c (Gr-1) (1:200, BioLegend, San Diego, CA, USA) following the manufacturer’s instructions. Living cells were assessed by using 1 mg/mL Propidium iodide (PI). Analyses of fluorescence staining were performed with a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Data were collected and analyzed using Kaluza® for Gallios-Acquisition and Flow Analysis Software (Beckman Coulter). We collected at least 10,000 viable cell events per sample in each experiment.
Fitc conjugated anti mouse cd8a
Manufactured by BioLegend
Sourced in United States
The FITC-conjugated anti-mouse CD8a is a flow cytometry antibody that specifically binds to the CD8a molecule expressed on the surface of mouse cytotoxic T cells. It is used to identify and enumerate CD8+ T cells in mouse samples.
Automatically generated - may contain errors
Lab products found in correlation
Apc conjugated anti mouse cd45, by BioLegend (1 mentions)
Apc conjugated anti mouse cd3e antibody, by BioLegend (1 mentions)
Apc conjugated anti mouse cd4, by BioLegend (1 mentions)
Apc cy7 conjugated anti mouse cd11b, by BioLegend (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscope lsm700, by Zeiss (1 mentions)
Vector trueview autofluorescence quenching kit, by Vector Laboratories (1 mentions)
Apc cy7 conjugated anti mouse cd45, by BD (1 mentions)
Fitc conjugated anti mouse cd11b, by BioLegend (1 mentions)
Anti mouse cd16 32 antibody clone 93, by BioLegend (1 mentions)
Apc conjugated anti mouse gr1, by BioLegend (1 mentions)
Pe conjugated anti mouse cd4, by BD (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Cell activation cocktail with brefeldin a, by BioLegend (1 mentions)
Facsdiva software, by BD (1 mentions)
Pe conjugated anti mouse cd3, by BioLegend (1 mentions)
Macsquant vyb flow cytometer, by Miltenyi Biotec (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
4 protocols using fitc conjugated anti mouse cd8a
1
Isolation and Characterization of Murine Splenocytes
2
Quantitative Analysis of Tumor-Infiltrating CD8+ and CD103+ T Cells
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Tumors removed from mice were rapidly deep-frozen using a slush containing dry ice and 2-methylbutane (lab-honeywell Cat.M32631) for 10 min and stored at −80°C. Frozen tumor sections were cut with 8 μm thickness in the cryostat at −25°C and fixed with cold acetone for 15 minutes, air-dried, and kept at −20°C for further use. Sections were rehydrated with 1X PBS for 5 min at RT two times, incubated in blocking buffer (3% BSA in 1× PBS) for 1 h at RT, and stained with FITC-conjugated anti-mouse CD8a (Biolegend, Clone:53–6.7, Cat:100706) and APC-conjugated anti-mouse CD103 (Biolegend, Clone:2E7, Cat:121413) at 1:100 dilution in blocking buffer overnight at +4°C, protected from light. Sections were then washed five times with 1× PBS for 5 min at RT followed by removal of autofluorescence using the Vector®TrueVIEW® Autofluorescence Quenching Kit (Vector Laboratories, Cat: SP-8400). Afterward, the nuclear stain was performed with 5 μg/ml Hoechst (Molecular Probes Invitrogen Hoechst 33342) in 1× PBS for 10 min at RT, slides were washed three times with 1X PBS for 5 min at RT and mounted with VECTASHIELD Vibrance Antifade Mounting Medium and stored at +4°C. All sections were imaged using LSM 700 laser confocal scanning microscope (Zeiss, Germany).
3
Flow Cytometry Immunophenotyping Assay
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Single-cell suspensions were incubated with anti-mouse CD16/32 antibody (clone 93; Biolegend, San Diego, CA) for 5 minutes prior to staining for immune cell markers for 15 minutes at room temperature. The following monoclonal antibodies were used: APC-CY7 conjugated anti-mouse CD45 (BD Biosciences Cat:557659 Clone:30-F11), FITC conjugated anti-mouse CD11b (Biolegend Cat:101205 Clone:M1/70), APC conjugated anti-mouse Gr1(Biolegend Cat:108412,Clone: RB6-8C5), FITC conjugated anti-mouse CD8a(Biolegend Cat:100706 Clone:53-6.7), PE conjugated anti-mouseCD4(BD Biosciences Cat:553048 Clone: RM4-5), APC conjugated anti-mouse PD-L1(Biolegend Cat:124311 Clone: 10F.9G2), and IFN-γ (XMG1.2).The flow cytometry analyses were performed using a BD Fortessa Flow Cytometer (BD Fortessa). BD FACS Diva software V.5.0.1 (BD) or Flow Jo (Tree Star) was used for data processed. For cytokine staining, harvested cells were incubated in RPMI-1640 medium with cell activation cocktail with brefeldin A (Bio legend) for 6 hours at 37°C, and stimulated cells were stained as described above.
4
MERS-CoV Antigen-Specific T Cell Response
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Splenocytes were prepared as described above. Splenocytes were added to 96-well plates (1 × 10⁶ cells/well) and stimulated for 6 h with MERS-CoV N-specific peptide (at 8 µg peptide/mL RPMI 1640 medium) in presence of the protein transport inhibitor Brefeldin A (Biolegend, San Diego, CA, USA; 5 µg/mL). Non-stimulated cells served as a background control and cells stimulated with 5 ng/mL PMA and 500 ng/mL ionomycin or with F2L peptide (8 µg/mL RPMI 1640 medium) were used as positive controls. After stimulation, cell surface antigens were stained using PE-conjugated anti-mouse CD3 (clone: 17A2, Biolegend, San Diego, CA, USA), PE/Cy7-conjugated anti-mouse CD4 (clone: GK1.5, Biolegend, San Diego, CA, USA), or FITC-conjugated anti-mouse CD8a (clone: 5H10-1, Biolegend, San Diego, CA, USA) antibody and incubated for 30 min on ice. The surface-stained cells were washed with staining buffer (MACS QuantTM Running Buffer, Miltenyi Biotec), then fixed and permeabilized with Fixation- and Perm/Wash-Buffer (Biolegend, San Diego, CA, USA), and finally stained for intracellular IFN-γ expression using APC-conjugated anti-mouse-IFN-γ antibody (clone: XMG1.2, Biolegend, San Diego, CA, USA) for 30 min on ice. Following final washes, cells were resuspended in staining buffer and analyzed using the MACS Quant® VYB flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany).
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