Primary TL cells were isolated from dissected murine TLs and cultured in RPMI-1640 medium with 10% FBS and 1% antibiotics/antimycotics. Freshly isolated or sequentially expanded TL cells were used for cell surface marker staining. The following fluorochrome-conjugated primary antibodies (BD Biosciences, San Jose, CA, USA) were used: PE-Cy7-CD3e, PE-CD4, and APC-CD8a. A FACSCalibur flow cytometer (BD Biosciences) with CellQuest Software (BD Biosciences) was used for data acquisition. Live cells were gated based on forward and side scatter. Data analyses were done using FCS Express 4 software package (De Novo Software, Los Angeles, CA, USA).
Cd8a apc
Manufactured by BD
Sourced in United States
CD8a-APC is a fluorescent-conjugated antibody that binds to the CD8a surface antigen. It is commonly used in flow cytometry applications for the identification and quantification of CD8-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 pe, by BD (3 mentions)
Cd4 fitc, by BD (2 mentions)
Cd45 percp cy5, by BD (2 mentions)
Canto 2, by BD (2 mentions)
Cd11b apc cy7, by BD (2 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (2 mentions)
Fcs express 4, by De Novo Software (1 mentions)
Pe cy 7 cd3e, by BD (1 mentions)
Fluorochrome conjugated primary antibodies, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Cd45 pe, by BioLegend (1 mentions)
Percp cy5.5 cd4, by BioLegend (1 mentions)
Hyaluronidase type 5, by Merck Group (1 mentions)
Lympholyte m gradient, by Cedarlane (1 mentions)
Collagenase a, by Merck Group (1 mentions)
Ammonium chloride, by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Facscanto, by BD (1 mentions)
Isotype control, by InvivoGen (1 mentions)
10 protocols using cd8a apc
1
Murine T Lymphocyte Isolation and Characterization
2
Liver Cell Isolation and Characterization
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Liver cells were prepared by passing through a 70 μm Falcon™ Cell strainer (Life sciences, MA, USA) and centrifugation of the supernatant into 40% Percoll (GE Healthcare, UK). The isolated cells were labeled with fluorophore-conjugated antibodies against PE-CD45 (BioLegend, CA, USA), FITC-CD3ε (BD Pharmingen, CA, USA), APC-CD8a (BD Pharmingen), and PerCP/Cy5.5-CD4 (BioLegend) for 30 min at 4 ℃. Labeled cells were assayed using a Gallio™ Flow Cytometer (Beckman Coulter, FL, USA). Data were analyzed using the FlowJo software (TreeStar, CA, USA).
3
Tumor-Infiltrating Lymphocyte Profiling
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Mice were sacrificed on the 14th day of the experiment. Tumors were collected for flow cytometric analysis; single-cell suspension was obtained using a digestion mix (0.5 mg/mL collagenase A, Sigma Aldrich; 0.2 mg/mL hyaluronidase type V, Sigma Aldrich; 0.02 mg/mL DNase I, Roche; per 0.25 g of tumor tissue). Red blood cells were lysed using 0.15 M ammonium chloride (Sigma Aldrich). Dead cells were removed by centrifugation using Lympholyte-M gradients (Cedarlane, Ontario, Canada). To identify the subpopulations of T lymphocytes, the following antibodies were used: PE-Cy7TM-CD3e, PE-CD4 and APC-CD8a (BD Pharmingen, catalog number: 558431, component: 51-9000790). The titers of antibodies was performed in accordance with the manufacturer’s instructions. Finally, to identify the level of NK cells, an anti-mouse CD49b (pan-NK cells) antibody was used (1 µg/106 cells; eBioscences, catalog number: 17-5971-82). In flow cytometric analyses (BD FACSCanto, BD), gate dividing negative from positive cells was based on isotype antibody control probes: PE-Cy™7 Hamster IgG1κ, PE and APC Rat IgG2aκ (BD Pharmingen, catalog number: 558431, component: 51-9000792) or APC Rat IgM (1 µg/106 cells, eBioscences, catalog number: 17-4341-82)30 (link). The titers of antibodies was performed in accordance with the manufacturer’s instructions.
4
Immunomodulation of Tumor Microenvironment
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CpG ODN 1826 (5ʹ-tccatgacgttcctgacgtt-3ʹ) and isotype control were purchased from InvivoGen (San Diego, CA, USA). Anti-OX40 (CD134) monoclonal antibody (clone OX86) and isotype control were purchased from BioXCell (Lebanon, NH, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin solution, phosphate-buffered saline (PBS) and TRIzol reagent were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The Matrigel was purchased from Corning (Corning, NY, USA). The TUNEL Apoptosis Detection Kit was purchased from KeyGen BioTECH (Nanjing, Jiangsu, China). The T Cell Isolation Kit was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Antibodies including CD45-PerCP-Cy5.5, CD3e-BV510, CD4-FITC, CD25-PE, OX40-APC, CD8a-FITC, CD11b-BV421, Gr-1-APC, F4/80-PE, CD8a-APC, CD44-PE-Cy7, CD62L-PE, IFN-γ-PE, and FoxP3-eFluor450 were purchased from BD Biosciences (San Jose, CA, USA) and Biolegend (San Diego, CA, USA).
5
Flow Cytometry Immunophenotyping Protocol
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For the flow cytometry assay, all cell suspensions of each of NALT, PN, CN and IN were adjusted to 1 × 106 cells per 1 mL in PBS. For analysis of the surface phenotype T cells were stained with CD4 (PerCP) and CD8a (APC) antibodies (BD biosciences). Cells were incubated for 30 min in the dark at room temperature. Subsequently, the cells were washed with PBS and fixed with 1% p-formaldehyde.
For the analysis of the percentage of B cells and IgG and IgA plasma cells, the antibodies (BD biosciences) were used: CD19 PE, CD138 APC and IgA FITC and IgG FITC. The cells were stained according to the BD Bioscience protocol for the detection of intracellular staining. The signal intensities were measured and analyzed by means of a FACSAria flow cytometer (Becton Dickinson) for the performance of relative fluorescence. 20 000 events were collected in the FSC/SSC point diagram.
For the analysis of the percentage of B cells and IgG and IgA plasma cells, the antibodies (BD biosciences) were used: CD19 PE, CD138 APC and IgA FITC and IgG FITC. The cells were stained according to the BD Bioscience protocol for the detection of intracellular staining. The signal intensities were measured and analyzed by means of a FACSAria flow cytometer (Becton Dickinson) for the performance of relative fluorescence. 20 000 events were collected in the FSC/SSC point diagram.
6
Antigen-Specific CD8+ T Cell Expansion
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Six to eight week old C57BL/6 female mice purchased
from the Jackson Laboratory were used in all immunization studies.
To assess the ability of iPEMs to expand antigen-specific CD8+ T cells during successive immunizations, mice were immunized
by intradermal (i.d.) injection with iPEM capsules (n = 6), a soluble mixture of SIINFEKL/polyIC (n =
6), or left untreated (n = 6). Both capsule groups
and vaccines formulated as soluble mixtures were prepared using the
same dose of antigen (60 μg) and polyIC (240 μg). Mice
were injected on day 0 and boosted with identical treatments at day
15 and day 28. For quantification of SIINFEKL-specific T cells, MHC-I
SIINFEKL tetramer (MBL International Corporation) staining was conducted
on peripheral blood every 7 days. Briefly, peripheral blood was collected
into ETDA-coated tubes and treated twice with ACK Lysing buffer (Thermo
Scientific) followed by a 1 mL PBS wash.1 (link) Cells were blocked with CD16/32 (BD Biosciences) for 10 min followed
by MHC-I tetramer (PE, H2-Kb, SIINFEKL) staining for 30
min. After tetramer staining, cell were stained with CD8a (APC, BD
Biosciences) for 20 min and washed twice with FACS buffer. DAPI was
added for viability assessment prior to analysis by flow cytometry
(CantoII, BD).
from the Jackson Laboratory were used in all immunization studies.
To assess the ability of iPEMs to expand antigen-specific CD8+ T cells during successive immunizations, mice were immunized
by intradermal (i.d.) injection with iPEM capsules (n = 6), a soluble mixture of SIINFEKL/polyIC (n =
6), or left untreated (n = 6). Both capsule groups
and vaccines formulated as soluble mixtures were prepared using the
same dose of antigen (60 μg) and polyIC (240 μg). Mice
were injected on day 0 and boosted with identical treatments at day
15 and day 28. For quantification of SIINFEKL-specific T cells, MHC-I
SIINFEKL tetramer (MBL International Corporation) staining was conducted
on peripheral blood every 7 days. Briefly, peripheral blood was collected
into ETDA-coated tubes and treated twice with ACK Lysing buffer (Thermo
Scientific) followed by a 1 mL PBS wash.1 (link) Cells were blocked with CD16/32 (BD Biosciences) for 10 min followed
by MHC-I tetramer (PE, H2-Kb, SIINFEKL) staining for 30
min. After tetramer staining, cell were stained with CD8a (APC, BD
Biosciences) for 20 min and washed twice with FACS buffer. DAPI was
added for viability assessment prior to analysis by flow cytometry
(CantoII, BD).
7
Flow Cytometry Analysis of Immune Cells After BMT
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For flow cytometry analyses at day +17 after BMT, peripheral blood, spleen and lymph nodes were harvested as described previously. [16 ] Single cell suspensions were stained with rat anti-mouse antibodies from BD Biosciences as follows: CD3e-APC-Cy7 (1:100), CD4-PE-Cy7 (1:800), CD8a-APC (1:200), CD25-PerCP-Cy5.5(1:200), CD11b-APC-Cy7 (1:400), B220-PerCP-Cy5.5 (1:100) or Nk1.1-PerCP-Cy5.5 (1:200). For chimerism analysis of blood and BM, antibodies against PE- Ly9.1 (1:100) and FITC-H2kb (1:50) were used. Regulatory T cell staining in blood and spleen were performed using Anti-Mouse/Rat FoxP3 Staining Set APC (eBioscience, San Diego, CA, USA) following the manufacturer´s instructions. Samples were analyzed by BD FACS Canto II (BD Biosciences) and FlowJo 7.6.5 Software (TreeStar Inc., Ashland, OR, USA).
8
Ovarian Cell Isolation and Characterization
9
Comprehensive Immune Cell Profiling
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The following anti-mouse antibodies were used for flow cytometry: Live/Dead Fixable Near IR Dead Cell Stain (Life Technologies), CD45-PerCP-Cy5.5 (BD Biosciences), CD3-PE (BD Biosciences), CD44-PE (BD Biosciences), CD8a-APC (BD Biosciences), CD4-APC (BD Biosciences), Foxp3-PE (BD Biosciences), CD11b-PE-Cy7 (BD Biosciences), F4/80-APC (BD Biosciences), Ly6C-PE (BD Biosciences), Ly6G-FITC (BD Biosciences), and Ly6G-APC-Cy7 (BD Biosciences). Flow cytometry was performed with Canto II (BD) and the data were analyzed with FlowJo 7.6 software (TreeStar). Live/dead cell discrimination was performed using Live/Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies). All of cell surface or intracellular stainings were done according to the manufacturer's instructions. T effector cells (Teff) were phenotyped as CD45+CD8+CD44+, regulatory T cells (Tregs) as CD45+CD4+Foxp3+, macrophages as CD45+CD11b+F4/80+, tumor-associated macrophages (TAMs) as CD45+CD11b+F4/80+CD206+, monocytic myeloid-derived suppressor cells (Mo-MDSCs) as CD45+CD11b+Ly6GlowLy6Chigh, and granulocytic myeloid-derived suppressor cells (G-MDSCs) as CD45+CD11b+Ly6GhighLy6Clow.
10
Multiparameter Flow Cytometry of Lymphocytes
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Lymphocytes from local lymph nodes were collected and stained with different fluorescence-labeled antibody cocktails as we previously described [27 (link)] to detect the percentages of CD4+ T cells (CD3-FITC, CD4-PE), CD8+ T cells (CD3-FITC, CD8a-APC), IFN-γ secreting cells (CD3-FITC, CD8-APC, IFN-γ-PE-Cy5-5), Tfh (T follicular helper) cells (CD45-APC-Cy7 (BD Pharmingen, San Diego, CA, USA), CD4-FITC (BD Pharmingen, USA), CD185-APC, PD-1-Pacific Blue (BD Pharmingen, USA)), germinal center (GC) B cells (B220-APC-Cy7, CD45-APC-Cy7 (BD Pharmingen, USA), CD95-PE, GL-7-APC), and plasma cells (B220-Pacific Blue, CD27-PE-Cy7, CD138-PE). Except where indicated, the remaining antibodies were purchased from BioLegend, San Diego, CA, USA. The stained cells were assessed by flow cytometry (BD LSRFortessa TM Cell Analyzer, San Jose, CA, USA), followed by FlowJo software analysis.
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