Mice were injected intradermally with 2.5ug mRNA(HA)-LNP or PBS at the upper left site of the back. Two weeks later, the same spot was injected with 2.5ug mRNA(eGFP)-LNP-DiI. Two days post injection, the mice were sacrificed and the skin and skin draining lymph nodes (axillary and brachial) were harvested. Single-cell suspensions were stained with fixable viability dye (Thermo Fisher), anti-CD45 (30 F11), CD11b (M1/70), Ly-6G (1A8), CD64 (X54–5/7.1), CD11c (N418) and MHCII (M5/114.15.2), as we previously described [71 (link)]. The antibodies were purchased from BioLegend, BD Biosciences and Tonbo Biosciences.
Fixable viability dye
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Fixable Viability Dye is a fluorescent dye used to identify live and dead cells in flow cytometry applications. It binds to proteins on cell surfaces, allowing distinguishing between viable and non-viable cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (37 mentions)
Facscanto 2, by BD (24 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (23 mentions)
Ionomycin, by Merck Group (23 mentions)
Facsdiva software, by BD (18 mentions)
Phorbol myristate acetate (pma), by Merck Group (16 mentions)
Lsrii flow cytometer, by BD (15 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (13 mentions)
Golgiplug, by BD (13 mentions)
Facsaria, by BD (13 mentions)
Lsrfortessa flow cytometer, by BD (12 mentions)
Cytofix cytoperm kit, by BD (11 mentions)
Fortessa, by BD (11 mentions)
Cytofix cytoperm, by BD (10 mentions)
Facscanto, by BD (10 mentions)
Facsymphony, by BD (9 mentions)
Cd11b, by BioLegend (8 mentions)
Facslyric, by BD (8 mentions)
Brilliant stain buffer, by BD (8 mentions)
Fc block, by BD (8 mentions)
414 protocols using fixable viability dye
1
Intradermal mRNA-LNP Vaccine Tracking
2
NK Cell Cytotoxicity Assay
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Purified human NK cells (105) were mixed with K562 cells at a ratio of 1:1 in V-bottomed 96-well plates, centrifuged at 100g for 1 min, and incubated 30 min, 1 h, 2 h or 3 h at 37 °C at 5% CO2. Negative controls were NK cells cultured alone and positive controls were NK cells cultured with IL-15 (100 ng/mL; Peprotech).
At indicated time points, the cells were harvested, stained with a fixable viability dye (ThermoFisher Scientific) and then surface-stained with anti-CD3 (clone SK7, 1/10; BD Biosciences), and anti-CD56 (clone NCAM16.2, 1/10; BD Biosciences) antibodies. The cells were subsequently fixed, permeabilised (Lysefix/PermIII® fixation/permeabilisation kit; BD Biosciences) and stained with anti-phospho-S6 ribosomal protein Ser 235/236 (clone D57.2.2E, 1/50; Cell Signaling Technology, Leiden, The Netherlands) or anti-PAkt S473 (clone M89-61, 1/40; BD Biosciences) antibodies.
Sample acquisitions were made on an ImageStream X Mark II (Amnis-EMD Millipore, Darmstadt, Germany) with ×40 magnification and analysed with IDEAS software (v6.0).
At indicated time points, the cells were harvested, stained with a fixable viability dye (ThermoFisher Scientific) and then surface-stained with anti-CD3 (clone SK7, 1/10; BD Biosciences), and anti-CD56 (clone NCAM16.2, 1/10; BD Biosciences) antibodies. The cells were subsequently fixed, permeabilised (Lysefix/PermIII® fixation/permeabilisation kit; BD Biosciences) and stained with anti-phospho-S6 ribosomal protein Ser 235/236 (clone D57.2.2E, 1/50; Cell Signaling Technology, Leiden, The Netherlands) or anti-PAkt S473 (clone M89-61, 1/40; BD Biosciences) antibodies.
Sample acquisitions were made on an ImageStream X Mark II (Amnis-EMD Millipore, Darmstadt, Germany) with ×40 magnification and analysed with IDEAS software (v6.0).
3
Cytokine Production Assay in Tumor Microenvironment
4
Thymic Epithelial Cell Immunophenotyping
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Thymic epithelial cells were acquired as described above. Cells were incubated with 2.4G2 before staining with other antibodies. The antibodies used included anti-CD45 (30-F11, eBioscience), anti-EpCAM (G8.8, Biolegend), anti-Ly51 (6C3, Biolegend), FITC labelled Ulex europaeus agglutinin-1 (UEA-1; Vector Laboratory), anti-IA/IE (M5/114.15.2, Biolegend), anti-SSEA-1 (MC-480, Biolegend), anti-LTβR (eBio3C8, eBioscience). C-CPE (C. perfringens enterotoxin) was produced as described47 (link) and biotin-conjugated; Streptavidin APC-eFluor 780 (eBioscience) was used for visualization of C-CPE. For intracellular staining of Ki-67 (B56, BD) and active caspase 3 (C92-605, BD), cells were fixed and permeabilzated with BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (554714) and stained according to the manufacture’s protocols. Fixable viability dye (L-34967, ThermoFisher) was used to exclude dead cells. For thymocyte and splenocyte analysis, cells were stained with anti-CD4 (RM4-5, eBioscience), anti-CD8 (53-6.7, eBioscience), anti-Va2 (B20.1, eBioscience), anti-Vb5 (MR9-4, eBioscience), and anti-CD24 (M1/69, Biolegend) before flow cytometry analysis. The samples were analyzed on BD LSRFortessa and FlowJo software (Tree Star Inc).
5
Neutrophil Metabolic and Functional Assays
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Neutrophils (0.3 × 106) were activated with Zy/op (100 µg/mL) for 1 h, at 37 °C and 5% CO2. Neutrophils were washed in PBS and incubated with MitoSOX (5 µM, Thermo, M36008) or Mitotracker Red (50 nM, Thermo, M7512) and Green (100 nM, Thermo, M7514) for 30 min. Samples were washed twice in PBS and immediately analysed by flow cytometry.
For glucose uptake assay, neutrophils (0.5 × 106) in glucose-free RPMI medium supplemented with the fluorescent glucose analogue 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG, Invitrogen, 30 µM, N13195) were activated with Zy/op (100 µg/mL) for 30 min, at 37 °C and 5% CO2. Neutrophils were washed twice in PBS and immediately analysed by flow cytometry.
To evaluate the expression of surface antigens, neutrophils were incubated with specific antibodies to GLUT1 (1:200, Abcam, Cat# ab209449), Ly6G (1:200, BD Bioscience, Cat# 560599) CD15 (1:100, BD, Cat# 562370) or CD11b (1:200, Biolegend, Cat# 101212) or the appropriate isotype controls for 1 h. Viable cells were assessed by incubating cells with Fixable Viability Dye (Thermo).
The fluorescence of samples was measured in a flow cytometer (FACSVerse™, BD Biosciences) and analysed using FlowJo software (Tree Star). Ten thousand cells were analysed52 (link).
For glucose uptake assay, neutrophils (0.5 × 106) in glucose-free RPMI medium supplemented with the fluorescent glucose analogue 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG, Invitrogen, 30 µM, N13195) were activated with Zy/op (100 µg/mL) for 30 min, at 37 °C and 5% CO2. Neutrophils were washed twice in PBS and immediately analysed by flow cytometry.
To evaluate the expression of surface antigens, neutrophils were incubated with specific antibodies to GLUT1 (1:200, Abcam, Cat# ab209449), Ly6G (1:200, BD Bioscience, Cat# 560599) CD15 (1:100, BD, Cat# 562370) or CD11b (1:200, Biolegend, Cat# 101212) or the appropriate isotype controls for 1 h. Viable cells were assessed by incubating cells with Fixable Viability Dye (Thermo).
The fluorescence of samples was measured in a flow cytometer (FACSVerse™, BD Biosciences) and analysed using FlowJo software (Tree Star). Ten thousand cells were analysed52 (link).
6
Comprehensive Immune Cell Profiling
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Prior to fluorochrome staining, FcRIII/II blocking was performed using the TrueStain fcX™ antibody (Biolegend, London, UK). Cell surface staining was done with anti-CD3 (clone 145-2C11), anti-CD4 (clone GK1.5), anti-CD8 (clone 53–6.7), anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD19 (clone 6D5), anti-CD26 (clone H194–112), anti-CD45 (clone 30-F11), anti-CD69 (clone H1.2F3), anti-CD172a (clone P84), anti-CD206 (clone C068C2), anti-EpCAM (clone G8.8), anti-F4/80 (clone BM8), anti-Ly6C (clone HK1.4), anti-Ly6G (clone 1A8), anti-MHC-I (clone AF6–88.5), anti-MHC-II (clone AF6–120.01), anti-NK1.1 (clone PK136), anti-PD-1 (clone 29F.1A12), anti-PD-L1 (clone 10F.9G2), anti-CD86 (clone GL-1), anti-CD40 (clone 3/23), anti-XCR1 (clone ZET; all BioLegend, London, UK) and anti-CD204 (clone 2F8, Biorad, Munich, Germany) antibodies, and Fixable Viability Dye (Thermo Fisher Scientific, Karlsruhe, Germany) was used to exclude dead cells. The gating strategy is depicted in Additional file 1 : Figure S1. Intracellular staining was done for arginase-1 (Polyclonal Sheep IgG; R&D Systems, Minneapolis, USA) using the eBioscience™ FoxP3/Transcription Factor Staining Buffer Kit (Thermo Fisher Scientific, Karlsruhe, Germany). Data were acquired on a BD LSRFortessa system (BD Bioscience, Heidelberg, Germany) and analyzed with FlowJo X software (FLOWJO LLC, Ashland, OR, USA).
7
Multiparameter Immune Cell Profiling
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PBMCs were stained with the following reagents and antibodies: fixable viability dye (Thermo Fisher), CD45 (30 F11), CD11b (M1/70), Ly-6G (1A8), CD64 (X54-5/7.1), CD11c (N418) and MHCII (M5/114.15.2). The antibodies were from BioLegend, BD Biosciences and Tonbo Biosciences.
8
Multi-parameter Flow Cytometry Analysis of Immune Cell Subsets
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Single-cell suspension was obtained as previously described.15 (link) The cells were stained with the following antibodies: anti-CD3e (145–2 C11), anti-CD8α (53–6.7), anti-CD11b (M1/70), anti-CD11c (N418), anti-CD25 (PC61.5), anti-F4/80 (BM8), anti-CD45 (30-F11), anti-Ly-6G (1A8), anti-Ly-6C (HK1.4), anti-CD31 (390), anti-I-A/I-E (M5/114.15.2), anti-ratIgG2aκ (RTK2758) and anti-ratIgG2bκ (RTK4530) from Biolegend or anti-CD4 (RM4-5), anti-CD69 (H1.2F3), anti-Klrg1 (2F1), anti-CD80 (16–10A1) and anti-NK1.1 (PK136) from ThermoFisher Scientific. Cells were also stained with Fixable Viability Dye (Thermo Fisher Scientific) and dead cells were gated out from the analysis. The stained cells were analyzed with flow cytometry (FACSLyric, BD Biosciences) and Flowjo software (BD Biosciences). Cell types were determined as following; CD8 T cells: CD45+/ CD3+/ CD8+, CD4 T cells: CD45+/ CD3+/ CD4+, NK cells: CD45+/ CD3-/ NK1.1+, DC1: CD45+/ CD11b-/ CD11c+, DC2: CD45+/ CD11b+/ CD11c+, EC: CD45-/ CD31+, CC: ssc high/ fsc high/ CD45-, MDSC: CD45+/ CD11b+/ Ly6G+ or Ly6C+, TAN: CD45+/ CD11b+/ Ly6G+, TAM: CD45+/ CD11b+/ F4/80 +.
9
Intranasal Delivery of Lipid Nanoparticles
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Mice were anesthetized by intraperitoneal injection with a mixture of Xylazine/Ketamine. The LNPs were given intranasally at the doses of 2.5, 5 and 10 μg in 30 μL sterile PBS by placing droplets gently on the left nostril and allowed the mice to inhale. The clinical performances of the mice were scored daily for 8 days as described previously (Shrum et al., 2014 (link)). Besides, the body weight was also measured daily. Some of the mice from the 10 μg LNP dose and corresponding PBS controls were sacrificed at indicated time points post-inoculation, and lung samples were harvested for histology and flow cytometry. For histology, the samples were fixed in 4% PFA overnight and then embedded in OCT. Eight micrometer thick sections were prepared using a cryostat and counterstained with DAPI. Stitched confocal pictures were taken using a Nikon A1 microscope. The lung samples for flow cytometry were digested using the collagenase/hyaluronidase technique also used for the skin samples (Kashem and Kaplan, 2018 (link)). The resulting single-cell suspensions were stained with the following markers: fixable viability dye (Thermo Fisher), MHC-II, CD11b, CD11c, CD24, CD45, CD64, Ly6G and Ly6C (All from BioLegend, except CD45 was from BD) (Yu et al., 2016 (link)).
10
Profiling Antigen-Specific Lymphocyte Responses
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At day 7 (peak of T cell responses; (Moon et al., 2009 (link))) and 14 post-injections (peak of B cell responses; (Pape et al., 2011 (link))), the mice were sacrificed and the skin draining lymph nodes (axillary, brachial and inguinal) harvested. Single-cell suspensions were generated using mechanical disruption through cell strainers. The mRNA-LNP platform does not require the use of T cell tetramers or fluorochrome-labeled antigen for B cells to study antigen-specific responses. There is only one antigen that the T and B cells react to, and the response is so robust that no need for magnetic enrichment either. Therefore, the cells were stained with either Tfh cell or B cell panels. The Tfh cell panel contained: fixable viability dye (Thermo Fisher), CD4 (GK1.5), CD44 (IM7), CD45 (OX-7), CD62L (H1.2F3), CD69 (H1.2F3), CXCR5 (L138D7), PD-1 (29F.1A12) and Bcl-6 (K112–91) from BioLegend and BD Biosciences. The gating strategy can be found in Suppl. Figure 3A . The B cell panel consist of dump (fixable viability dye, F4/80, CD11b), CD38 (90), B220 (RA3–6B2), CD138 (281–2), GL-7 (GL-7), Sca1 (D7), IgD (11–26c.2a) and IgM (RMM-1). The gating strategy is presented in Suppl. Figure 3B . The stained samples were run on LSRFortessa™ (BD Biosciences) and the resulting data analyzed with FlowJo 10.
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