Surface marker staining was performed after the FcR block using Human TruStain FcX Fc Receptor blocking solution (BioLegend). Cells were incubated with the Live/Dead Fixable Yellow Dead Cell Stain Kit (Life Technologies). Surface marker-stained cells were analyzed on BD LSRFortessa with FACSDiva software (BD Biosciences). The gating strategy of the FACS analysis was shown in Supplemental Fig. 13 . The following antibodies were used for FACS staining: anti-CD45RA-FITC (clone HI100), anti-CD25-PE (BC96), anti-4-1BB-BV421 (4B4-1), anti-CD8-BV510 (RPA-T8), anti-CD103-BV605 (Ber-ACT8), anti-CD4-BV711 (OKT4), anti-Tim-3-APC (F38-2E2), anti-CD3-Alexa Fluor 700 (UCHT1), anti-CD15-BV510 (W6D3), anti-CD11b-BV605 (M1/70), anti-CD45-BV786 (HI30), anti-CD14- Alexa Fluor 700 (HCD14), and IgG1 isotype control (MOPC-21) purchased from Biolegend. Anti-ICOS-PerCP eFluor 710 (ISA-3) and IgG1 isotype control (P3.6.2.8.1) were purchased from eBioscience. Anti-OX-40-PE-CF594 (ACT35), anti-PD-1-PE Cy7 (EH12.1), and anti-CCR3-PE-CF594 (5E8) were purchased from BD Bioscience.
Live dead fixable yellow dead cell stain kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LIVE/DEAD Fixable Yellow Dead Cell Stain Kit is a laboratory reagent used to identify dead cells in a sample. It contains a fluorescent dye that binds to dead cells, allowing for their detection and separation from live cells during analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (9 mentions)
Dnase 1, by Merck Group (9 mentions)
Facsdiva software, by BD (8 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (7 mentions)
Anti cd16 32 clone 93, by BioLegend (6 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (4 mentions)
Lsrfortessa x 20, by BD (4 mentions)
Lsrfortessa flow cytometer, by BD (4 mentions)
Collagenase a, by Roche (4 mentions)
Cyan adp, by Beckman Coulter (3 mentions)
Lsr fortessa cytometer, by BD (3 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (3 mentions)
Facsdiva, by BD (3 mentions)
Collagenase 4, by Merck Group (3 mentions)
Lsrii flow cytometer, by BD (3 mentions)
Cell activation cocktail, by BioLegend (3 mentions)
Siglecf e50 2440, by BD (3 mentions)
Lsrii system, by BD (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Igg1 isotype control mopc 21, by BioLegend (2 mentions)
99 protocols using live dead fixable yellow dead cell stain kit
1
Comprehensive Immune Cell Profiling by Flow Cytometry
2
Multicolor Flow Cytometry Staining
3
Isolation and Characterization of Tumor-Infiltrating T Cells from Ovarian Cancer
4
Splenocyte Stimulation and T Cell Analysis
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Splenocytes were harvested on day 16–18 post B16-F10 tumor inoculation and plated for culture. The splenocytes were re-stimulated with TDPAs isolated from B16F10 lysates or neoantigens (Actn4, Eef1, Tubb3, Dag1) for 72 hours. After stimulation, splenocytes were washed and stained. Single cell suspensions were blocked with Fc Block for 5 minutes on ice and then stained with anti-mouse CD3, CD8, CD4 (Supplementary Table 1 ). Live/dead fixable yellow dead cell stain kit (Life Technologies) was applied for live/dead cell discrimination. For surface staining, samples were first incubated with Fc block for 5 min on ice and stained with anti-mouse CD3, CD8, CD4 (Supplementary Table 1 ). Cells were then fixed, permeabilized, and stained for intracellular IFN-γ. All flow cytometric analysis was done using a Beckman Coulter CyAn ADP and analyzed using software Summit 5.2. The data were presented as the percentage of CD8+IFN-γ+ in CD8+ cells, and the percentage of CD4+IFN-γ+cells in CD4+ cells. Differences were compared and the overall P value was calculated by Mann Whitney test using the GraphPad Prism 5.0. (P value: *, P<0.05; **, P<0.01; ***, P<0.005.) The representative plots of relative abundance IFN-γ production T cells were showed in Supplementary Fig. 10 .
5
Comprehensive NK Cell Immunophenotyping
6
Comprehensive Characterization of PLGA Nanoparticles
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PLGA (AP059; LA:GA=50:50 (w:w); MW: 45,000–55,000 Da), mPEG-PLGA (AK037; LA:GA=50:50 (w:w); MW: ~25,000 Da), PLGA-PEG-NH2 (AI058; MW: ~17,000 Da), PLGA-PEG-Mal (AI052; LA:GA=75:25; MW: ~63,400 Da), and Poly(lactide-co-glycolide)-Rhodamine B (PLGA-Rb) (AV011; LA:GA=50:50; Mn=10,000–30,000 Da) were obtained from Polyscitech®. All other chemicals were obtained from Sigma-Aldrich unless otherwise noted.
Collagenase/Hyaluronidase and Bovine Pancreas DNase I-PBS solution were obtained from Stemcell Technologies. LIVE/DEAD® Fixable Yellow Dead Cell Stain Kit and ACK lysis buffer were obtained from Life Technology. Recombinant Murine IL-2 was obtained from PeproTech. αPD-1 (clone: RMP1-14) was from BioXcell. The peptides Actn4 (NHSGLVTFQAFIDVMSRETTDTDTADQ), Eef2 (FVVKAYLPVNESFAFTADLRSNTGGQA), Tubb3 (FRRKAFLHWYTGEAMDEMEFTEAESNM), or Dag1 (TAVITPPTTTTKKARVSTPKPATPSTD) were obtained from peptide 2.0 (Chantilly, VA). All antibodies used for flow cytometric assays are listed in theSupplementary Table 2 .
Collagenase/Hyaluronidase and Bovine Pancreas DNase I-PBS solution were obtained from Stemcell Technologies. LIVE/DEAD® Fixable Yellow Dead Cell Stain Kit and ACK lysis buffer were obtained from Life Technology. Recombinant Murine IL-2 was obtained from PeproTech. αPD-1 (clone: RMP1-14) was from BioXcell. The peptides Actn4 (NHSGLVTFQAFIDVMSRETTDTDTADQ), Eef2 (FVVKAYLPVNESFAFTADLRSNTGGQA), Tubb3 (FRRKAFLHWYTGEAMDEMEFTEAESNM), or Dag1 (TAVITPPTTTTKKARVSTPKPATPSTD) were obtained from peptide 2.0 (Chantilly, VA). All antibodies used for flow cytometric assays are listed in the
7
Flow Cytometric Analysis of gB RNA Transfection
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BHK cells were electroporated with 0.1 μg of FL-gB RNA along with 4.2 μg of mouse thymus RNA. Following overnight culture (16–18 h) the cells were detached using Trypsin-EDTA (Invitrogen Inc.). Cells were initially stained for viability using a LIVE/DEAD Fixable Yellow Dead Cell Stain Kit (Life Technologies) and were then stained for surface expression of gB with either ITC88 (1 μg per sample), 1G2 (1 μg per sample) or none (as control), followed by goat anti-human IgG (H+L) Alexa Fluor 647 (0.25 μg per sample; Cat no. A-21445, Life Technologies). Cells were fixed and permeabilized, and then stained with an anti-dsRNA mAb (0.25 μg per sample; clone J2, Cat no. 10010500, English and Scientific Consulting Bt) that had been conjugated using a Zenon R-Phycoerythrin Mouse IgG2a Labeling Kit (Life Technologies). All samples were analysed by flow cytometry on an LSR II instrument (BD Biosciences).
8
Bovine PBMC Immunophenotyping by Flow Cytometry
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Total PBMC were enumerated and 1×106 cells were aliquoted per well. Cells were washed once with FACS buffer (0.3% BSA, 0.9% sodium azide, PBS). All antibodies were diluted in FACS buffer and cells were incubated on ice for 20 minutes and washed twice in FACS buffer. The anti-bovine antibodies used are outlined in Table 1 and Table 2 . To determine cell viability either LIVE/DEAD Fixable Yellow Dead Cell Stain Kit or LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Life Technologies, Grand Island, NY) were used. Prior to staining cells with antibodies, cells were incubated with LIVE/DEAD stain for 30 minutes on ice and washed twice with FACS buffer. The emission of LIVE/DEAD Fixable Yellow Dead Cell Stain allowed us to detect its fluorescence using the Violet laser, Quantum-Dot 525 channel on the LSR-II (BD Biosciences, San Jose, California), whereas LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit was detected using the Red laser, APC-Cy7 channel. FACS DIVA Software was used for acquisition and analysis (BD Biosciences, San Jose, California). FlowJo Software 9.6.4 Version (TreeStar, Ashland, USA) was also used for analysis.
9
Isolation and Characterization of Tumor-Infiltrating T Cells from Ovarian Cancer
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Stage III-IV human ovarian carcinoma tumors and malignant ascites fluid were procured through Surgical Pathology at Weill Cornell Medicine and Memorial Sloan Kettering Cancer Center. The Weill Cornell Medicine IRB conducted a review of the project described and determined that the activities do not constitute human subjects research as these specimens were classified as surgical discard and remained totally de-identified. Collection and analysis of OvCa specimens at Memorial Sloan Kettering Cancer Center were approved by an institutional IRB protocol and were obtained under informed consent. Tumor single-cell suspensions were prepared as previously described28 (link). Malignant ascites samples were centrifuged for 10 min at 1,300rpm. Ascites supernatants were collected, depleted of cells by passing through 0.22μm filters, and stored frozen at −80°C as in small aliquots until use. Red blood cells in cell pellets were lysed with ACK lysing buffer (Gibco). Tumor infiltrating T cells (CD45+CD20−CD14−CD3+ CD4+ or CD8+) were sorted from tumor single cell suspensions or malignant ascites using a BD FACSAria II SORP cell sorter at Flow Cytometry Core Facility in Weill Cornell Medicine. Dead cells were excluded using the LIVE/DEAD Fixable Yellow Dead Cell Stain Kit (Life Technologies). All OvCa specimens used and analyzed in this study are described in Supplementary Table 1 .
10
Splenocyte Culture and Cytokine Analysis
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Example 14
Splenocytes were harvested on day 16-post tumor inoculation and plated for culture. The splenocytes were re-stimulated with tumor antigen as previously prepared for 72 hours. The supernatant was collected for cytokine analysis with ELISA. Splenocytes were washed and stained. Live/dead fixable yellow dead cell stain kit (Life Technologies) was applied for live/dead cell discrimination. For surface staining, samples were first incubated with Fc block for 5 min on ice and stained with anti-mouse CD3, CD8, CD4 (Table 1). Cells were then fixed, permeabilized, and stained for intracellular IFN-γ. All flow cytometric analysis was done using a Beckman Coulter CyAn ADP and analyzed using software Summit 5.2. The data were presented as the percentage of CD8+IFN-γ+ in CD8+ cells, and the percentage of CD4+IFN-γ+ cells in CD4+ cells. Differences were compared and the overall P value was calculated by analysis of unpaired t test using the GraphPad Prism 5.0. (P value: *, P<0.05; **, P<0.01; ***, P<0.005). The representative plots of relative abundance IFN-γ production T cells were shown in
Scheme 1 depicts a convergent synthetic route for the preparation of a PLGA cleavage peptide-PEG as described herein.
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