Cryopreserved TILs were thawed in warmed RPMI plus benzonase (MilliporeSigma) and washed in RPMI. Cells were plated in a round-bottom 96-well plate at 1–2e6 cells/well. Cells were washed with PBS, blocked with mouse CD16/32 Fc block (BioLegend), and then stained with Zombie UV dye (BioLegend) and surface antibody cocktail, then permeabilized with eBioScience FoxP3 Fixation/Permeabilization kit (eBioscience, Waltham, MA, USA) according to manufacturer’s protocol. They were stained with intracellular antibody cocktail, then fixed with 2% paraformalin and kept on ice until acquisition on a BD Symphony A3 (BD). Data were compensated for fluorescent spillover and analyzed using FlowJo 10.8.1 (RRID: SCR_008520 ) (BD).
Symphony a3
Manufactured by BD
Sourced in United States
The Symphony A3 is a laboratory equipment designed for precision measurement and data analysis. It features advanced technology to provide accurate and reliable results. The core function of the Symphony A3 is to perform analytical tasks required in various scientific and research settings.
Automatically generated - may contain errors
Lab products found in correlation
Ghost dye alexa fluor 700, by Cytek Biosciences (2 mentions)
Cytofix, by BD (2 mentions)
Cd3 fitc, by BD (2 mentions)
Cd8 pe, by BD (2 mentions)
Cd4 apc, by BD (2 mentions)
Zombie uv dye, by BioLegend (1 mentions)
Benzonase, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Golgiplug, by BD (1 mentions)
Diva software, by BD (1 mentions)
Macs column, by Miltenyi Biotec (1 mentions)
Hyclone, by Cytiva (1 mentions)
Cytofix cytoperm plus kit, by BD (1 mentions)
Cd45ra bv 421, by BD (1 mentions)
Lysis solution, by BD (1 mentions)
Sc 377530, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
15 protocols using symphony a3
1
Comprehensive Immunophenotyping of Cryopreserved TILs
2
Isolation and Cytokine Profiling of CD8+ T Cells
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Spleen cells were obtained from two mice from each of infected HLA-A2.1-transgenic and WT mouse groups at 4-5 weeks after infection and suspended in Hank’s balanced salt solution (HyClone [cytiva], Mariborough, MA) with 2% fetal bovine serum (Sigma-Aldrich). The spleen cells were pooled within the same experimental group, and CD8+ T cells were purified from these spleen cells using magnetic beads conjugated anti-mouse CD8α (clone 53-6.7) monoclonal antibodies (mAbs) (Miltenyi Biotech, Auburn, CA) and MACS column (Miltenyi) (17 (link)). The purified CD8+ T cells were then stimulated with 5 ng/ml phorbol myristate acetate (Sigma) and 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of Golgi Plug (BD Biosciences, Mountain View, CA) as previously described (24 (link), 28 (link)). After the stimulation, the cells were incubated with APC-conjugated anti-CD8α mAb (clone 53-6.7) in combination with FITC-conjugated mAbs to 15 different T cell receptor (TCR) Vβ chains, followed by fixation and permeabilization with Cytofix/Cytoperm Plus kit (BD Biosciences) and stained with PE-conjugated anti- IFN-γ (XMG1.2) or isotype control (R3-34) mAbs (24 (link)). All mAbs were purchased from BD Biosciences. Cells were analyzed on BDSymphony A3 using DIVA software (BD Biosciences).
3
Multiparameter Immune Cell Profiling
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After being thawed, PBMCs were washed with warm RPMI followed by PBS. The cells were then stained with viability marker Ghost dye-Alexa Fluor 700 (Tonbo Biosciences) for 30 minutes at 4 °C, then washed with FACS buffer (1X PBS supplemented with 1% bovine serum albumin and .1% sodium azide). To prevent unintended antibody binding to Fc receptors, cells were blocked with FcR blocking reagent, human (Miltenyi Biotec) for 10 minutes at 4 °C. The cells were then stained at a 1:50 dilution with antibodies to immune cell targets as follows: CD3-FITC (BD Biosciences, 555332), CD8-PE (BD Biosciences, 555635), CD4-APC (BD Biosciences, 555349), CCR7-buv395 (BD Biosciences, 568681), CD45RA-bv421 (BD Biosciences, 569620) for 30 minutes at 4 °C. The cells were then washed twice with FACS buffer and fixed using cytofix from BD Biosciences for 30 minutes at 4 °C, followed by a wash with FACS buffer and resuspension in FACS buffer. Cell populations were acquired using the BD Symphony A3, and Flow v10.8 software was used for analysis. Fluorescence-minus-one controls were utilized to identify cell populations, and each experiment had a single-stained control and negative control.
4
PBMC Immunophenotyping by Flow Cytometry
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PBMCs were unthawed and washed with warm RPMI then washed with PBS. Cells were then stained with live/dead marker Ghost Dye-Alexa Fluor 700 (Tonbo Biosciences) for 30 min at 4°C according to the manufacturer’s instructions, then washed with FACS buffer. Human FcR blocking reagent, human (Miltenyi Biotec) was used to block unwanted binding of antibodies to FC receptors for 10 min at 4°C. Cells were then stained for immune cell markers CD3-FITC (BD Biosciences, Cat#: 555332), CD8-PE (BD Biosciences, Cat#: 555635), CD4-APC (BD Biosciences, Cat#: 555349) for 30 min at 4°C. Stained cells were fixed with cytofix (BD Biosciences) for 30 min at 4°C, then washed and suspended in FACS buffer (PBS with 1% BSA and 0.1% sodium azide). BD Symphony A3 was used to acquire cell populations. Analysis was conducted with Flow v10.8 software. Fluorescence-minus one controls were used to identify cell populations, and each experiment had single-stained and no-stain controls.
5
Monocyte Immunophenotyping with Amylin
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Each blood sample (100 µl) was incubated with a mixture of CD14 (Abcam, ab203294, 1:400), and human amylin (Santa Cruz, SC-377530, 1:100) primary antibodies for 30 min at ambient temperature. Blood was lysed in 2 ml of lysis buffer (1× BD lysis solution, Cat# 349202, BD Biosciences) for 5 min and was washed in 1×PBS with 4% fetal calf serum (FCS). The remaining cells after washing were incubated in secondary antibodies for 30 min at ambient temperature, then were washed and resuspended in 0.5 ml of 1×PBS with 4% FCS. Secondary antibodies were Alexa Fluor 488 goat anti Mouse IgG (H + L) (cat# A11029; Invitrogen, dilution 1:200 in 1×PBS with 4% FBS) for human amylin, and Alexa Fluor 568 goat anti Mouse (cat# A11004, Invitrogen, dilution 1:200, in 1xPBS with 4% FBS), for CD14, respectively. Flow analysis was done on Cytometers BD Symphony A3. The remaining cells were gated on SSC-A vs. FSC-A to determine live cell populations. Doublets (aggregated cells) were gated out using FSC-H vs. FSC-A. Non-aggregated cells were further gated to select monocytes that were positive for CD14 and/or human amylin. Negative control (no antibody) and positive control (human monocytes) were used to set the upper and lower boundaries (Supplemental Fig. S4 ).
Data was acquired using BDFacsDiva and analyzed using FlowJo v10 software.
Data was acquired using BDFacsDiva and analyzed using FlowJo v10 software.
6
Chronic PD-1 Blockade Alters Brain T Cells
7
Monitoring Myeloid Differentiation by Flow Cytometry
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HL60, DMSO-treated HL-60, THP-1, and PMA-treated THP-1 cells were seeded at 1.5E5 cells/well on a clear 96-well plate. The cells were washed with 200 µL/well of PBS without Ca2+ and Mg2+, supplemented with 2% FBS (FACS buffer). For HL60 cells, CD11b-FITC (BDPharmingen; Cat: 562,793 Clone: ICRF44) was added to each well at 1:40 dilution (50 µL) in FACS buffer and incubated at RT, being covered from light, for 15 min. For THP-1 cells, a combination of CD11b and CD14-APC/Cy7 stains (Cat: 301,820 Clone: M5E2) were used to monitor CD11b upregulation in addition to macrophage differentiation. The cells were washed twice with FACS buffer at 1400 rpm for 5 min. each and LIVE/DEAD Fixable Near-IR Dead Cell stain (ThermoFisher Scientific, Waltham, MA, USA) was added to each well at a dilution of 1:500 (50 µL) and incubated on ice, covered from light, for 15 min. The cells were washed again and resuspended in 200 µL FACS buffer. Fluorescence measurements were acquired using either Guava flow cytometer (EMD Millipore, Burlington, MA, USA) or they were acquired at a Symphony A3 (BD Biosciences, Franklin Lakes, NJ USA). Data was analyzed with FlowJo software V10. Induction was considered to be successful if CD11b expression in viable cells was found to be ~70% or higher.
8
Quantifying Cellular Signaling Pathways
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For cell surface and intracellular labeling, single-cell suspensions were stained with fluorochrome-labeled antibodies listed in the Supplementary Data (Table S1) . Activation of signaling pathways was evaluated by intracellular staining of pAKT (as mTORC2 substrate), p4EBP1 (as mTORC1 substrate), pSRC (as proximal T cell receptor activation substrate), and pSTAT5 (as IL2-receptor substrate) after 4 days of drug exposure. Flow cytometry data were acquired with LSRII or Symphony A3 cytometers (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo software (TreeStar, Ashland, OR, USA).
9
Annexin V/PI Assay for Cell Death
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The types of cell death were evaluated using Annexin V/Propidium iodide (PI) staining, according to the manufacturer’s instructions. SNU-478 cells (2 × 105 cells/well) were plated in 6-well cell culture plates. After 24 h of incubation, the cells were co-incubated with AMG (5 μM) and/or gemcitabine (500 nM) for 48 h. After collecting the cells, they were reacted with Annexin V (BioLegend, San Diego, CA, USA) and propidium iodide (PI) (Invitrogen, Waltham, MA, USA). The samples were acquired using fluorescence-activated cell sorting (FACS) Symphony A3 (BD Biosciences, San Diego, CA, USA), and results were analyzed with the FlowJo Software v. 10.8.1 (TreeStar, San Carlos, CA, USA).
10
Multiparametric Flow Cytometry and Sorting
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