All antibodies were purchased from Biolegend (San Diego, CA) unless noted otherwise. FcR blocking was performed with anti-CD16/32 antibody prior to staining with various combinations of the following antibodies: anti-CD4 (RM4–5), -CD25 (PC61), -Foxp3 (FJK-16s) (eBioscience, San Diego, CA) -CD80 (16–10A1) -CD86 (GL-1), -I-A/I-E (M5/114.15.2). Viability was assessed with fixable violet dead cell stain kit (Invitrogen, Carlsbad, CA) or 4′,6-Diamidino-2-Phenylindole, Dilactate (DAPI) (Biolegend). Foxp3 staining was performed with the Foxp3 Staining Buffer Set according to the manufacturer’s protocol (eBioscience). Flow cytometric data were collected using a Beckman Coulter CyAn ADP Analyzer. Analysis was performed using FlowJo software (FlowJo, Ashland, OR).
Fixable violet dead cell stain kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Fixable Violet Dead Cell Stain Kit is a laboratory product designed to identify and distinguish between live and dead cells in a sample. It contains a violet-fluorescent dye that binds to dead cells, allowing for their detection and quantification using flow cytometry or other fluorescence-based analysis techniques.
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Lab products found in correlation
Anti cd45 buv395, by BD (3 mentions)
Lsrfortessa, by BD (3 mentions)
Macsquant, by Miltenyi Biotec (3 mentions)
Foxp3 transcription factor staining buffer kit, by Thermo Fisher Scientific (3 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (2 mentions)
Apc anti cd8a, by Thermo Fisher Scientific (2 mentions)
Anti cd4 apc efluor 780, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Perm wash buffer, by BD (2 mentions)
Symof1, by Thermo Fisher Scientific (2 mentions)
Anti mouse human irf4 3e4, by Thermo Fisher Scientific (2 mentions)
Anti mouse bcl6 k112 91, by BD (2 mentions)
4 6 diamidino 2 phenylindole dilactate dapi, by BioLegend (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Cyan adp analyzer, by Beckman Coulter (1 mentions)
Phosflow fix buffer, by BD (1 mentions)
Tumor necrosis factor (tnf), by RayBiotech (1 mentions)
Il 1β, by Aviscera Bioscience (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Pd 1 clone j105, by Thermo Fisher Scientific (1 mentions)
20 protocols using fixable violet dead cell stain kit
1
Multiparametric Immune Cell Analysis
2
PBMC Signaling Pathway Activation Assay
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PBMC were incubated with live/dead fixable violet dead cell stain kit (Invitrogen, L34955) for 30 minutes. Cells were then treated with either 100 ng/ml TNF (RayBiotech, 230-00243-50), LPS (Sigma Aldrich, L2880) or IFN-α2b (GenScript, 203002-50), or 1 µg/ml IL-1β (Aviscera Bioscience, 00756-01-59), for up to 60 minutes. Cells were fixed with BD Phosflow Fix Buffer (BD Biosciences, 557870), permeabilized with BD Perm Buffer III (Biosciences, 558050) and stained with the following antibodies: PE anti-phospho-STAT1 (pY701) (BD Biosciences, 612564); Alexa Fluor® 488-anti-phospho-STAT3 (pY705) (Becton Dickinson 557814); or PE anti-NF-κB phospho-p65 (pS529) (BD Biosciences, 558423) and quantified by flow cytometry as described above.
3
Quantifying C. rodentium Infection by Flow
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Cells were stained for flow cytometry using the following fluorescence-conjugated antibodies diluted in FACS Buffer (2% FBS, 0.01% Sodium Azide, PBS): PE anti-CD326 (EpCAM) (Clone: G8.8, eBioscience), BUV395 anti-CD45.2 (Clone: 104, BD Biosciences), 488 anti-GFP (Clone; FM264G, BioLegend). Dead cells were excluded with the Fixable Violet Dead Cell Stain Kit (Invitrogen). Samples were acquired on the BD LSRFortessa and analyzed with FlowJo Software (Treestar). The geometric mean fluorescence intensity (MFI) for GFP-C. rodentium expression was assessed and the background MFI determined in uninfected controls was subtracted from infected samples.
4
Phenotypic Characterization of TIL Infusion
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A small fraction of TIL infusion products was cryopreserved for subsequent phenotypic characterization by flow cytometry. In brief, after thawing, cells were rested for 24 hours, and then stained using antibodies directed against: CD3 (clone SK7), CCR7 (clone 150503), CD28 (clone CD28.2), CD62L (clone SK11) (all BD Biosciences), CD4 (clone S3.5), CD8 (clone 3B5), CD45RA (clone MEM-56), CD27 (clone CLB-27/1) (all Invitrogen) and PD-1 (clone J105, eBioscience). Fixable Violet Dead Cell Stain Kit (Invitrogen) was used to exclude dead cells. Cells were analyzed on a LSR2 SORP flow cytometer (BD Biosciences) and data was processed using Flowjo_V10 software.
5
Cell Staining and Flow Cytometry
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Cells were centrifuged and transferred to conical bottom plate (Greiner Bio-One, 650101), stained for 20 min in cold PBS with Fixable Violet Dead Cell Stain Kit (1/4000, Invitrogen, L34955) and washed in FACS Buffer (PBS 0.5% BSA 2 mM EDTA). Extracellular staining was performed in FACS buffer for 30 min on ice. After staining, cells were washed in FACS buffer and stained with the corresponding secondary antibody. Finally, cells and compensation beads (eBioscience, 01-1111-42) were acquired with MACS Quant (Miltenyi) flow cytometer and data were analyzed with FlowJo software.
6
Cytokine Production Assay of TILs
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A total of 2×105 TILs were cultured with 2×105 cells from tumor digest or tumor cell lines, in the presence of 1 µL/mL Golgiplug (BD Biosciences). After 5 hours of incubation, cells were stained using anti-CD8 antibody (clone SKI, BD Biosciences) and live/dead marker (Fixable Violet Dead Cell Stain Kit, (Invitrogen)). Intracellular cytokine production was detected after fixation and permeabilization with the Cytofix/Cytoperm kit (BD Biosciences) and antibodies against interferon-γ (IFN-γ) (clone B27), IL-2 (clone Mq1-17H12), and tumor necrosis factor α (TNF-α) (clone Mab11), all from BD Biosciences. Cells were analyzed on an LSR2 SORP flow cytometer and data were processed using Flowjo_V10 software.
7
Detailed Cytometry Staining Protocol
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Cells were centrifuged and transferred to conical bottom plate (Greiner Bio-One, 650101), stained for 20 min in cold PBS with Fixable Violet Dead Cell Stain Kit (1/4000, Invitrogen, L34955) and washed in FACS Buffer (PBS 0.5% BSA 2 mM EDTA). Extracellular staining was performed in FACS buffer for 30 min on ice. For surface marker expression of human cells, antibodies anti-CD28, anti-TCRα/β, anti-CD3, anti-CD4, and anti-HA were used and are described in Supplementary Table 1 . After staining, cells were washed in FACS buffer and fixed with Cytofix/Cytoperm (BD Biosciences, 554714).
For intracellular cytometry, cells were washed twice in Perm/Wash buffer (BD, 554723), and incubated for 1 h in ice with anti-LAT or anti-CD45. In the next step, cells were washed twice with perm/wash followed by staining with the correspondent secondary antibody (see Supplementary Table1 ).
Finally, cells and compensation beads (eBioscience, 01-1111-42) were acquired with BD FACS Verse and MACS Quant (Miltenyi) flow cytometer and data were analyzed with FlowJo software.
For intracellular cytometry, cells were washed twice in Perm/Wash buffer (BD, 554723), and incubated for 1 h in ice with anti-LAT or anti-CD45. In the next step, cells were washed twice with perm/wash followed by staining with the correspondent secondary antibody (see Supplementary Table
Finally, cells and compensation beads (eBioscience, 01-1111-42) were acquired with BD FACS Verse and MACS Quant (Miltenyi) flow cytometer and data were analyzed with FlowJo software.
8
Multiparametric Flow Cytometry Analysis
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Cell samples from each time point from the VOR in vitro single- or multiple q24h-dose simulation studies (see above) were washed with DPBS and stained with the fixable violet dead cell stain kit (Invitrogen, Carlsbad, CA) for 20 min. Cells were then stained with an extracellular antibody cocktail in fluorescence-activated cell sorting (FACS) buffer (PBS + 5% FBS) containing anti-CD3-fluorescein isothiocyanate (FITC) clone 1F4, anti-CD4-PE clone W3/25, and CD45-PeCy7 clone OX-1 (BioLegend, San Diego, CA) for 30 min at room temperature. Cells were fixed with 4% paraformaldehyde and kept at 4°C until the last time point was collected. Fixed samples were washed and permeabilized using the eBioscience Foxp3/transcription factor staining buffer set (Invitrogen, Carlsbad, CA) and stained with the anti-H4 K5/8/12/16 antibody clone 3HH4-4C10 (Millipore, Burlington, MA) conjugated to AF647 for 30 min at room temperature. Samples were analyzed on the LSR II (BD Biosciences, San Jose, CA) and in FlowJo (version 10).
9
Fe-doped Nanodiamond Synthesis and Characterization
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Fe-doped nanodiamond (FeND), with an average grain size of 3.5–6.0 nm, was purchased from Ray Technologies, Ltd., Israel. This diamond is synthesized through detonation, then purified and Fe-doped. Fe can be doped onto the ND via various techniques such as ion implantation, doping, or surface decoration.26,27 The specifics of the process used for doping the ND with Fe have not been disclosed by the supplier. Human Serum Albumin (HSA), doxorubicin hydrochloride (DOX), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and hydrogen peroxide (H2O2) were bought from Sigma-Aldrich (USA). Tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was bought from Abcam, USA, and Fixable Violet Dead Cell Stain Kit was bought from Invitrogen, USA. All chemicals were used as received without further purification.
10
CD4+ Cell Depletion and IL-17A Neutralization
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