Samples from all time points were analyzed simultaneously. Cells were first washed in PBS and then blocked with human IgG (1 mg/mL, Sigma-Aldrich). The pDC-T cell fractions were stained with CD123-APC (eBioscience), and the aforementioned FcεR1-PE, CD3-APC-Cy7, CD80-PerCP-eF710, CD86-AF488, HLA-DR-eF450, and CD40-PE-Cy7. The mDC-T cell fractions were labeled with BDCA1-APC (eBioscience) and CD19-APC-Cy7 (Biolegend) along with the FcεR1-PE, CD3-APC-Cy7, CD80-PerCP-eF710, CD86-AF488, HLA-DR-eF450, and CD40-PE-Cy7. Samples were run on a BD FacsVerse (BD Biosciences, San Josa CA). pDCs were identified as CD123+ CD3−, and mDCs as BDCA-1+ CD3− CD19− cells. After gating on each cell type, the MFI of each activation marker and HLA-DR was determined. Appropriate isotype controls were included in each experiment.
Cd3 apc cy7
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CD3-APC-Cy7 is a fluorescent-conjugated antibody used in flow cytometry applications. It is designed to detect and quantify the expression of the CD3 cell surface antigen, which is a complex of glycoproteins that is expressed on the surface of T cells. The APC-Cy7 fluorophore is used for the labeling of the antibody, providing a specific emission spectrum that can be detected by flow cytometry instrumentation.
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Lab products found in correlation
Cd8 fitc, by Thermo Fisher Scientific (3 mentions)
Cd4 pe, by Thermo Fisher Scientific (3 mentions)
Cd3 apc, by Thermo Fisher Scientific (2 mentions)
Kc57 fitc antibody, by Beckman Coulter (2 mentions)
Ccr5 pe, by R&D Systems (2 mentions)
Ccr6 percp cy5, by BioLegend (2 mentions)
Cd90 alexa fluor 647, by BioLegend (2 mentions)
Ccr5 pe cy7, by BD (2 mentions)
Cd4 fitc, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (2 mentions)
Facscanto, by BD (2 mentions)
Cytofix cytoperm plus kit, by BD (2 mentions)
Facsverse, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Cd3 apc cy7, by BioLegend (1 mentions)
Cd19 apc cy7, by BioLegend (1 mentions)
Cd123 apc, by Thermo Fisher Scientific (1 mentions)
Human igg, by Merck Group (1 mentions)
Facsaria iiu, by BD (1 mentions)
Cd4 alexa fluor 488, by BioLegend (1 mentions)
10 protocols using cd3 apc cy7
1
Characterization of Dendritic Cell Subsets
2
HIV Infection Analysis by Flow Cytometry
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Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD8-FITC, CD4-FITC, CD4-PE, CD3-APC, CD3-APC-Cy7 (eBioscience, San Diego, CA), CCR5-PE-Cy7 (BD Biosciences, San Jose, CA), CCR5-PE (R&D Systems, Minneapolis, MN), CCR6-PerCp-Cy5.5, CD90 Alexa Fluor-647 (Biolegend, San Diego, CA). For the HIV-infection experiments, following infection for 6–7 days, intracellular levels of p24 were analyzed as described previously8 (link). Briefly, after surface staining, cells were washed, fixed and permeabilized (20 min) following instructions provided in the Cytofix/Cytoperm Plus kit (BD Biosciences) and stained for intracellular p24 with KC57-FITC antibody (Beckman Coulter; Danvers, MA) for 30 min. Analysis was performed on BD FACSCalibur or BD FACSCanto flow cytometers (BD Biosciences) using FACSdiva software, and data analyzed with FlowJo software (Tree Star, Inc. Ashland, OR). Expression of surface markers was measured by the percentage of positive cells and the mean fluorescence intensity (MFI).
3
Isolation and sequencing of activated CD4+ T cells
4
HIV Infection Analysis by Flow Cytometry
5
Assessing Monocyte Iron Dynamics
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PBMCs were isolated by Ficoll-Paque (GE Healthcare) and washed with 0.9% NaCl before incubation with 5 µM iron-quercetin (FeQ2) alone and/or in combination with 5 µM apoBet v1 in media containing neither phenol red nor FCS for 18 h as previously described [41 (link)]. Subsequently, cells were stained with combinations of calcein-AM (Thermo-Fisher, Waltham, MA, USA), CD3-APC-Cy7 (eBioscience, clone SK7), CD14-APC-Cy7 (Biolegend, clone M5EZ), HLADR-PE (Biolegend, San Diego, Calif, clone L243PC), and CD86-PE-CY7 (Biolegend, clone IT2.2) for flow cytometric analysis. calcein-AM violet was used as a living marker and to determine the labile iron load in living cells. Doublets were excluded before gating on CD3+ cells in the lymphocyte population or on CD14+ cells in the monocytic population. In monocytic cells this was followed by consecutive gating for HLADR+, CD86+ and calcein+ and geometric mean fluorescence intensity (MFI) was calculated for each fluorochrome. Acquisition and analyses were performed on a FACS Canto II machine (BD Bioscience, San Jose, CA, USA) using the FACSDiva Software 6.0 (BD Biosciences).
6
Milk and Soy Antigen Stimulation of PBMC
7
Naive T Cell Proliferation Assay
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Naïve T cells were purified from cryopreserved peripheral blood mononuclear cells (PBMCs) using the Naïve Pan T Cell Isolation Kit (Miltenyi Biotec). After purification, isolated naïve T cells were >99% CCR7+ CD45A+ as determined by flow cytometry with CD3‐APC‐Cy7 (Tonbo), CCR7‐PE‐Cy7 (Miltenyi Biotec), and CD45RA‐APC (Biolegend). Naïve T cells were stained with Cell Proliferation Dye eFluor‐670 (eBioscience) as recommended by the manufacturer. Purified mucosal CD1a+ or CD14+ cells (5 × 103 cells) were plated with naïve T cells (7.5 × 104 cells) (1:15 ratio) in round‐bottom 96‐well plates, in Xvivo 15 media (Invitrogen) supplemented with 10% human AB serum (Valley Biomedical). After 6 days in culture, proliferation of T cells was assessed by flow cytometry after staining with zombie yellow dye (Biolegend) and CD3‐APC‐Cy7, CD8‐FITC (Tonbo), CD4‐PE, CD103‐PE‐Cy7 (eBiosciences), and CD11c‐PerCp‐Cy5.5 (Biolegend). Naïve T cells alone were used as a negative control. For some experiments, TGFβ receptor 1 blocker, SB431542 (10 μmol/L, Tocris Cookson Inc) (Ochiel, Ochsenbauer, et al., 2010 ), was added to the cultures at the beginning of each experiment.
8
Phenotyping T Cell Responses
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PBMCs for TCR profiling were cultured in TCRPMI as described above and reported previously (12 (link)). For TCR overexpression experiments we used AIM V media. PBMCs were washed with PBS two times and once with media, subsequently resuspended at 5 × 105 cells/100 µL and aliquoted in a 96-well plate for a 12-h rest. Then, cells were stimulated with 20 µg/mL of antigenic peptide and 2 µg/mL of CD28/49d in 100 µL of media for 24 h. PBMCs were then washed with wash buffer as described above, but RNAsin Plus inhibitor was excluded. PBMCs were then stained with CD3-APCCy7 (Thermo Fisher, cat. no. 47-0036-42), CD8a-PE (Thermo Fisher, cat. no. 12-0088-42), CD4-PECy7, and CD137-APC (Biolegend, cat. no. 309810) antibody for 20 min. Subsequently, cells were washed, resuspended in wash buffer and 7-aminoactinomycin D (7-AAD) (BD, cat. no. 559925) or DAPI was added immediately prior to FACS analysis or sorting. Multimer staining was performed with tetramers as previously described, and MART-1 (ELAGIGILTV) HLA-A2 tetramer was made in-house (12 (link)). Tetramers for NY-ESO-1 (MBL, cat. no. TB-M011-1), CMV pp65 (MBL, cat. no. TB-0010-2), and EBV BMLF1 (MBL, cat. no. TB-M011-2) were purchased.
9
SARS-CoV-2 Antigen-Specific T-Cell Assay
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The polyclonal antigen-specific cellular assay was performed on thawed PBMC samples grown in supplemented RPMI1640 (R10), containing 1 µg/mL of SARS-CoV-2 PepMix™ (JPT Peptides, Berlin, Germany), for 48 h in a 37 °C, 5% CO2 chamber. After initial incubation, cells were harvested, washed with FACS buffer, centrifuged, and stained using a viability dye (Live/Dead Fixable Blue, Thermo Fisher Scientific, Waltham, MA, USA) followed by surface staining using human antibodies: CD3 APC-Cy7, CD4 BV421, CD8 BV605, CD38 PE-Cy7, CD19 BB700, CD27 BB515, CD134 (OX40) BV650, CD197 (CCR7) BV510, and CD45RA APC. All human antibodies were purchased from BD Biosciences, San Jose, CA, USA. Two types of compensation beads were used as compensation controls (UltraCompBeads, Invitrogen, Waltham, MA, USA; ArCTM Armine Reactive Compensation Bead kit, Invitrogen, Waltham, MA, USA) [32 (link)].
The samples were acquired using LSRFortessa (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software v.10.8 (BD Biosciences, San Jose, CA, USA). The gating strategy used for clustering memory B and T cells is represented inSupplementary Figure S1 . PBMCs grown in unsupplemented R10 were used as a negative control and as a measure of baseline fluorescence. The minimum percentage considered for final analysis was 1% in the gate of activated memory cells.
The samples were acquired using LSRFortessa (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software v.10.8 (BD Biosciences, San Jose, CA, USA). The gating strategy used for clustering memory B and T cells is represented in
10
FACS Analysis of Immune Cell Subsets
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Single cells isolated from spleen and intestinal lymph node were stained for FACS analysis as described previously (16 (link)). Lymphocytes were stained using the Zombie Violet™ Fixable Viability Kit (BioLegend Biotechnology Co., LTD, China) and were stained for surface markers including CD3-APC-Cy7 and CD8-APC (Thermo Fisher Scientific Co., LTD., USA); cells were then fixed and permeabilized by the Intracellular Fixation and Permeabilization Buffer Set (Thermo Fisher Scientific Co., LTD., USA) and stained for intracellular expression markers such as IFN-γ-PerCP and IL-4-PE (Thermo Fisher Scientific Co., LTD., USA). DCs were stained for surface markers including CD11c+-PE-Cy7, CD80-APC, CD86-FITC, and MHC-II-PE (Thermo Fisher Scientific Co., LTD., USA). Data were acquired with BD FACSVerse (Becton Dickinson Co., LTD., Canada) and analyzed with BD FACSuite (Becton Dickinson Co., LTD., Canada).
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