P14 CD8+ T cells were isolated and activated by pMHC liposomes or anti-CD3/anti-CD28 as described above. The media was changed and supplemented with 30 U/ml rhIL-2 after 48 hr. On day 5 post-activation, MC38 target cells were pulsed with GP33 peptide in OptiPRO SFM media (Gibco) for 1 hr at 37°C. Pulsed MC38 cells were washed 5 times with PBS, seeded in a 96 well plate at 6×105 cells/ml in T cell media, and incubated for 4 hr to allow adhesion. Activated P14 T cells were washed 5 times with PBS and incubated with MC38 cells at a 5:1 effector-to-target ratio overnight. For analysis of IFNγ expression, cells were washed with PBS, blocked with Fc Shield (Tonbo) for 10 minutes on ice, and stained with anti-mouse CD8 (Biolegend, Clone: 53–6.7) and anti-mouse IFNγ (Biolegend, Clone: XMG1.2) for 30 min on ice before collection on a BD Accuri C6.
Fc shield
Manufactured by Cytek Biosciences
The Fc Shield is a lab equipment product designed to reduce Fc receptor-mediated effects in flow cytometry applications. It functions by blocking Fc receptors, thereby minimizing non-specific binding and improving the specificity of antibody-based detection.
Automatically generated - may contain errors
Lab products found in correlation
Moflo xdp, by Beckman Coulter (2 mentions)
Novocyte 3001, by Agilent Technologies (2 mentions)
Zombie yellow live dead staining, by BioLegend (2 mentions)
Anti mouse ifn γ, by BioLegend (1 mentions)
Anti mouse cd8, by BioLegend (1 mentions)
Accuri c6, by BD (1 mentions)
Red blood cell lysis buffer, by BioLegend (1 mentions)
Zombie nir, by BioLegend (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Facscelesta, by BD (1 mentions)
4 protocols using fc shield
1
Quantification of Antigen-Specific CD8+ T Cell Cytotoxicity
2
Flow Cytometry Surface Staining and Sorting
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For surface marker staining, cells were blocked with Fc Shield (anti-mouse CD16/CD32, Tonbo) for 10min then incubated with antibodies of interest for 30 min. Samples were washed extensively with sterile FACS buffer (PBS, 2% FCS, 2mM EDTA) then analyzed using Novocyte 3001 (ACEA Biosciences). Cells were gated on singlets and dead cells were excluded using Zombie Yellow live/dead staining (Biolegend). Data were analyzed using FlowJo software (BD).
For cell sorting, samples were stained, washed, then sorted by Moflo XDP (Beckman Coulter) directly into 10% FCS containing complete RPMI media. Cells were washed once with complete RPMI media before culturing.
For cell sorting, samples were stained, washed, then sorted by Moflo XDP (Beckman Coulter) directly into 10% FCS containing complete RPMI media. Cells were washed once with complete RPMI media before culturing.
3
Splenic Cell Preparation for Flow Cytometry
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Tissues were prepared for flow cytometric analysis as previously described [42 (link)]. Briefly, spleens were harvested under sterile conditions and were homogenized by forcing the tissue through 100 μm cell strainers using the plunger from a syringe. Single-cell suspensions were prepared, and red blood cells were removed using red blood cell lysis buffer (BioLegend). The resulting suspension was passed through a 70 μm cell strainer and washed once with PBS. Cells were resuspended to a concentration of 0.5-1 × 106 cells/mL for flow cytometric analysis in FACS Buffer containing PBS, 5% fetal bovine serum, 1 mM ethylenediaminetetraacetic acid (EDTA) (Sigma Aldrich), and 0.1% sodium azide (Sigma Aldrich). Cell viability was measured by staining cell suspensions with ZombieNIR (BioLegend). Prior to surface staining, cells were incubated with Fc Shield (TonboBiosciences) for murine specimens. For surface staining of murine specimens, cells were stained in FACS buffer with the following antibodies: CD3 (145-2C11), CD4 (GK1.5), CD8 (53–6.7), CD11b (M1/70), Gr-1 (RB6-8C5) (all from BioLegend). Fluorochromes that overlapped with the emission spectra of PV-10 were not used in this study. Cells were acquired by FACS Celesta (BD Biosciences), and the data were analyzed with FlowJo software (Tree Star).
4
Flow Cytometry Surface Staining and Sorting
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