Mice were sacrificed seven days after immunization and splenocytes were isolated as previously described [6 (link)]. In brief, red blood cells were lysed using ACK buffer and then splenocytes were counted using a Z1 Coulter counter. For tetramer analysis, splenocytes were immediately blocked for Fc (Tonbo Bioscience) and stained for B8R tetramer (AF488, 1:300, NIH Tetramer Facility, Atlanta, GA) followed by staining with anti-CD3 (PeCy7, 1:100, clone 145-2C11) and anti-CD8α (eFlour450, 1:200, clone 145-2C11). Cells were then stored overnight at 4C in a 1:1 of IC Fixation Buffer (ThermoFisher Scientific) and FACS buffer. For analysis of cytokine production, 1.7x106 cells were plated in a 96 well dish and incubated for five hours in the presence of B8R20-27 (TSYKFESV) or OVA257-264 (SIINFEKL) peptides and brefeldin A (eBioscience). Splenocytes were then subjected to FC block (Tonbo Bioscience) and stained with anti-CD3 (FITC, 1:200, clone 145-2C11) and anti-CD8α (eFlour450, 1:200, clone 53–6.7) before treatment with fixing and permeabilization buffers (eBioscience). Cells were then further stained with anti-IFNγ (APC, 1:300, clone XMG1.2). Samples were acquired using the LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed with FlowJo software (Tree Star, Ashland, OR).
Fc block
Manufactured by Cytek Biosciences
The Fc block is a laboratory instrument designed to prevent non-specific binding of Fc-containing molecules, such as antibodies, to Fc receptors on cells. Its core function is to block Fc-mediated interactions, enabling more accurate and specific analysis of target molecules during flow cytometry or other immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Fixable viability dye, by Thermo Fisher Scientific (2 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (1 mentions)
Eflour450, by Thermo Fisher Scientific (1 mentions)
Live dead fixable violet dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Accucheck counting beads, by Thermo Fisher Scientific (1 mentions)
Fluorochrome conjugated antibodies, by Thermo Fisher Scientific (1 mentions)
Lsr fortessa, by Tree Star (1 mentions)
Fortessa, by BD (1 mentions)
Lsr 2, by BD (1 mentions)
Cd11b m1 70, by Cytek Biosciences (1 mentions)
Cx3cr1, by BioLegend (1 mentions)
Sirpα, by BioLegend (1 mentions)
H2 kb, by BD (1 mentions)
Tcrγ δ, by BioLegend (1 mentions)
Siglec f, by BD (1 mentions)
5 protocols using fc block
1
Splenocyte Isolation and Tetramer Analysis
2
Isolation of Adult Cardiac Cells
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Adult hearts were transcardially perfused with PBS containing 0.9 mM Ca2+, and atria and valves were removed. Single-cell suspensions were obtained as previously described (Pinto et al., 2016 (link)). Briefly, tissue was minced and incubated with collagenase type IV (600 U/mL, Worthington, LS004188) and dispase II (1.2 U/mL, Thermo Fisher, 17105041) in DPBS containing 0.9 mM Ca2+ for 45 min at 37°C and filtered through both a 40 μm and then a 30 μm filter to ensure a single-cell suspension. Yellow amine-reactive live/dead cell dye (Thermo Fisher, L34959) or LIVE/DEAD Fixable Violet Dead Cell Stain kit (Thermo Fisher, L34964) was used for live/dead discrimination. Cells were then incubated in Fc block (1:100, Tonbo Bioscience, 70–0161 U500) followed by indicated antibodies and fixed with 1% PFA in flow cytometry buffer. Primary antibodies used for flow cytometry are listed in Supplementary file 1f . Accucheck counting beads (Thermo Fisher, PCB-100) were added according to the manufacturer’s protocol prior to acquisition. Data were acquired on an LSRFortessa (BD Bioscience) and analyzed using FlowJo software version 10 (BD Bioscience). Cell counts were calculated according to the Accucheck counting bead manufacturer’s recommendations and normalized to the weight of individual ventricles.
3
Murine Tumor Cell Immunophenotyping
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Single cell suspensions prepared from mouse tumors and spleens were pre-incubated with 1 μl/ml anti-CD16/32 monoclonal antibody (Fc block; Tonbo) for 30 min at 4 °C and then stained with indicated fluorochrome-conjugated antibodies (eBioscience) and a fixable viability dye (Life Technologies). Labeled cells were acquired BD LSRFortessa and data processed using FlowJo software (Treestar).
4
Detailed Flow Cytometry Protocol
5
Immune Profiling of Tumor Microenvironment
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After tumors were harvested from individual mice, 2 cubic millimeters of tumor were separated from the whole tumor and were frozen in liquid nitrogen for gene expression analysis. The rest of the tumor tissue was processed into single cells by homogenizing through 40 μm nylon mesh strainers into complete media (RPMI+10% FBS). First, single cell samples were blocked with Fc block (Tonbo), and then incubated with fluorescently labeled antibodies to identify immune markers using murine specific antibodies against CD3, CD4, CD8, CD11b, CD19, CD45, CD11c, CD137, CD25, GITR, Lag3, Tim3, PD1, PDL1, MHCII, and CD86 (eBioscience, Thermo Fisher), and a fixable viability dye (Affymetrix, Thermo Fisher). Appropriate unstained and “Fluorescence Minus One” controls were used. Next, the cells were fixed, permeabilized, and stained with intracellular markers Ki67 and FoxP3 (eBioscience, Thermo Fisher). Labeled single cell suspensions of tissues were collected individually from each mouse and subjected to flow cytometry following a described gating strategy [34 (link)]. Samples were collected on a 4-laser Fortessa X-20 cytometer (BD Biosciences) and analyzed with FlowJo V10 software (Flowjo, LLC, Ashland, OR). The level of infiltration was determined using prism software.
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