To assess apoptosis, cells were stained using the Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) kit (Roche). PMN were fixed with Fix & Perm Medium A (Life Technologies) and permeabilized with nuclear permeabilization buffer before incubation with TUNEL reaction mixture according to manufacturer’s instructions. To assess mitochondrial membrane potential, cells were stained using 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide (JC-1) (BD Biosciences). Membrane depolarisation is characterized by a fluorescence emission shift from green (~529 nm, FL2) to red (~590, FL1). PMN (1 X 106 cells) were incubated with JC-1 for 15 min at 37°C and 5% CO2 according to manufacturer’s instructions. To determine intracellular p53 levels, PMN were fixed with Fix & Perm Medium A (Life Technologies) and permeabilized with nuclear permeabilization buffer before incubation with anti-p53 APC (MiltenyiBiotec) for 15 min at room temperature. All flow cytometric analysis was performed immediately with a BD FACSCanto II using FACS DIVA and FlowJo software.
Fix perm medium a
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fix & Perm Medium A is a solution used in the preparation of samples for microscopy analysis. It is designed to fix and preserve the structure and morphology of cellular and tissue specimens.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (4 mentions)
Fix perm medium b, by Thermo Fisher Scientific (3 mentions)
Live dead aqua, by Thermo Fisher Scientific (2 mentions)
Anti cd8 apc h7 sk1, by BD (2 mentions)
Anti cd26 fitc ba5b, by BioLegend (2 mentions)
Anti cd161 pe cy7 hp 3g10, by BioLegend (2 mentions)
Anti cd3 alexafluor700 ucht1, by BioLegend (2 mentions)
Anti ifn γ pe dazzle594 4s b3, by BioLegend (2 mentions)
Anti il 17 bv421, by BioLegend (2 mentions)
Anti cd4 bv711 okt4, by BioLegend (2 mentions)
Golgiplug, by BD (2 mentions)
Cd14 pe, by BioLegend (1 mentions)
Il 1β fitc, by Thermo Fisher Scientific (1 mentions)
Sa fitc, by BioLegend (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Anti human cd4 fitc, by Thermo Fisher Scientific (1 mentions)
Anti human il 17 pe, by Thermo Fisher Scientific (1 mentions)
Pharmingen, by BD (1 mentions)
Facscalibur cytometer, by BD (1 mentions)
11 protocols using fix perm medium a
1
Apoptosis, Mitochondrial Potential, and p53 Analysis
2
Evaluating Immune Responses to Extracellular Vesicles
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Healthy EVs with or without LL37 were used to stimulate healthy PBMCs for determining IP10, IL1β and IFNα positive cells. Healthy PBMCs were stimulated with EV or EV/LL37 complex for 6 h and 16 h in the presence of Brefeldin-A (5 µg ml–1, Biolegend, Cat#420601) for IL1β and IP10 analysis, respectively. Of note, Brefeldin-A was added at t = 0 and t = 6th hour of incubation for IL1β and for IP10, respectively. The cells were then fixed with Fix & Perm Medium A (Invitrogen, Cat# GAS001S100). Cells were first stained with CD14-PE (Biolegend, Cat#325606) and then to detect the level of intracellular IL1β or IP10 were stained wither with IL1β-FITC (e-Biosciences, Cat# 12-7018-81) or IP10-Biotin (BD, Cat# 555048) then SA-FITC (Biolegend, Cat# 405202) in the presence of saponin (0.1%).
IFNα secreting cells were analysed with Miltenyi Biotech’s (San Diego, CA, USA) IFNα Secretion Assay and Detection Kit (PE-labelled, Miltenyi, Cat# 130-094-161) from PBMCs in accordance with kit instructions. Additionally, in order to identify IFNα secreting pDCs, cells were stained with BDCA-2-FITC (Biolegend, Cat# 354208) and then analysed by BD Accuri C6 flow cytometer.
IFNα secreting cells were analysed with Miltenyi Biotech’s (San Diego, CA, USA) IFNα Secretion Assay and Detection Kit (PE-labelled, Miltenyi, Cat# 130-094-161) from PBMCs in accordance with kit instructions. Additionally, in order to identify IFNα secreting pDCs, cells were stained with BDCA-2-FITC (Biolegend, Cat# 354208) and then analysed by BD Accuri C6 flow cytometer.
3
Phenotyping of MAIT Cells from BAL and PBMCs
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When sufficient cells were available, freshly isolated matched BAL lymphocytes and PBMCs were simultaneously stimulated using PMA (25 ng/mL) and ionomycin (500 ng/mL) for 6 hours at 37°C in 96-well microplates. After 6 hours, plates were washed in phosphate buffered saline (PBS) before cells were stained with 1:500 of either MR1 6-FP (PE) or MR1 5-OP-RU (PE) tetramer (NIH Tetramer Core Facility). Tetramer staining was performed in the dark at room temperature for 40 minutes, followed by surface staining at 4°C for 20 minutes. The surface staining antibody master-mix included: 1:25 of anti-CD4 BV711 (OKT4, BioLegend), 1:100 of anti-CD8 APC-H7 (SK1, BD Biosciences), 1:50 of anti-CD26 FITC (BA5b, BioLegend), 1:50 of anti-CD161 PE-Cy7 (HP-3G10, BioLegend) and 1:800 of a fixable viability dye (Live/Dead Aqua, Invitrogen). Cells were then fixed (Fix & Perm Medium A, Invitrogen) at room temperature in the dark for 15 minutes and permeabilized (Fix & Perm Medium B, Invitrogen) for 20 minutes at 4°C. Intracellular antibodies were added during permeabilization and included 1:50 each of: anti-CD3 AlexaFluor700 (UCHT1, BioLegend), anti-IFN-γ PE-Dazzle594 (4S.B3, BioLegend), anti-granzyme B AlexaFluor647 (GB11, Biolegend), and anti-IL-17 BV421 (BL168, BioLegend). Cells were PBS washed, acquired using the LSRFortessa™ (BD Biosciences) and the data then analyzed using FlowJo (v10.4).
4
Multiparametric Analysis of mMDSCs
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Stimulated mMDSC were incubated with Fc Block for 15 min on ice and stained with fluorochrome-conjugated antibodies against 25F9, CD206, and CD163 on ice for 20 min. Cells were washed with PBS/2% BSA followed by Fix & Perm Medium A (Invitrogen, Carlsbad, CA, USA). Cells were washed again, re-suspended in PBS, and analyzed using an LSRFortessa (BD Biosciences, Franklin Lakes, NJ, USA).
5
Flow Cytometric Analysis of IL-17 Expression
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The stimulated cells were harvested, centrifuged, resuspended in PBS, and then incubated with 5 uL anti-human CD4-FITC (eBioscience) at room temperature in the dark for 20 minutes. After washing with fresh PBS twice, cells were incubated with 100 uL FIX & PERM medium A (Invitrogen) for 15 minutes. Following centrifugation and resuspension, cells were treated with 100 uL FIX & PERM medium B (Invitrogen) and incubated in the dark for 20 minutes. Then 5 uL anti-human IL-17-PE was added (eBioscience) to the suspension. A parallel control group was treated instead with 5 uL anti-mouse PE-anti-IgG1. Both suspensions were incubated at room temperature in the dark for 15 minutes. Cells were then washed, centrifuged, suspended in PBS, and then analyzed by flow cytometry.
6
CD152 Expression Analysis by Flow Cytometry
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For CD152 labelling (BD PharmingenTM, BD Biosciences, San Jose, CA, USA), 1 × 106 cells were washed with 1 mL PBS and incubated with Fix & Perm Medium A solution (InvitrogenTM, Carlsbad, CA, USA) for 15 min at room temperature in the dark for permeabilization of the cell membrane. The cells were then rewashed with PBS and incubated with Fix & Perm Medium B and CD152 (PE-Cy5) antibody for 30 min at room temperature in the dark. Afterwards, the cells were washed with PBS and fixed with 1% paraformaldehyde (PFA). Cell acquisition was performed using a FACS Calibur cytometer (BD Biosciences, San Jose, CA, USA), and the analysis was performed using FlowJo software (FlowJo, Ashland, TN, USA).
7
Multiparametric Flow Cytometry Phenotyping
8
Multiparameter Analysis of T Cell Responses
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When sufficient cells were available, freshly isolated matched BAL lymphocytes and PBMCs were simultaneously stimulated using PMA (25 ng/mL) and ionomycin (500 ng/mL) for 6 hours at 37°C in 96-well microplates. After 6 hours, plates were washed in phosphate buffered saline (PBS) before cells were stained with 1:500 of either MR1 6-FP (PE) or MR1 5-OP-RU (PE) tetramer (NIH Tetramer Core Facility). Tetramer staining was performed in the dark at room temperature for 40 minutes, followed by surface staining at 4°C for 20 minutes. The surface staining antibody master-mix included: 1:25 of anti-CD4 BV711 (OKT4, BioLegend), 1:100 of anti-CD8 APC-H7 (SK1, BD Biosciences), 1:50 of anti-CD26 FITC (BA5b, BioLegend), 1:50 of anti-CD161 PE-Cy7 (HP-3G10, BioLegend) and 1:800 of a fixable viability dye (Live/Dead Aqua, Invitrogen). Cells were then fixed (Fix & Perm Medium A, Invitrogen) at room temperature in the dark for 15 minutes and permeabilised (Fix & Perm Medium B, Invitrogen) for 20 minutes at 4°C. Intracellular antibodies were added during permeabilization and included 1:50 each of: anti-CD3 AlexaFluor700 (UCHT1, BioLegend), anti-IFN-γ PE-Dazzle594 (4S.B3, BioLegend), anti-granzyme B AlexaFluor647 (GB11, Biolegend) and anti-IL-17 BV421 (BL168, BioLegend). Cells were PBS washed, acquired using the LSRFortessa™ (BD Biosciences) and the data then analysed using FlowJo (v10.4).
9
NK Cell Activation by SARS-CoV Spike Proteins
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PBMC from one control were rested overnight with 10 ng/ml human recombinant IL-15 (R&D Sytems) and NK cells were then isolated using EasySep Human NK Cell Isolation Kit (Stemcell Technologies). Nunc MaxiSorp Flat-Bottom plates (ThermoFisher) were coated with 1.5 ng/well of SARS-CoV-1 or SARS-CoV-2 spike proteins and incubated at 4 C for 6 h, then washed and blocked with R10 for 19 h at 4 C. After blocking, 100 ul of vNARs (5ug/ml) were added to each well and plates were incubated 30 min at 37 C. Plates were then washed with PBS 4x, followed by addition of 25,000 purified NK cells per well. GolgiStop (BD Biosciences) and Brefeldin A (ThermoFisher) were also added at 1:1000, and CD107a APC (clone H4A3; BD Biosciences) was added 1:100 in R10. After 6 h at 37 C, cells were stained for surface markers Aqua Live/Dead stain (ThermoFisher), CD3 PE-Tx Red (clone 7D6; ThermoFisher), CD56 PE-Cy7 (clone NCAM16.2; BD Biosciences), CD19 AF700 (clone HIB19; BD Biosciences), and CD16 BUV496 (clone 3G8; BD Biosciences) in FACS Buffer for 10 min at RT. Cells were fixed with Fix & Perm Medium A (ThermoFisher) for 15 m at RT. Fluorescence was evaluated on a LSRII (BD Biosciences). Data was analyzed using FlowJo version 10.7.1. NK cells were gated as CD3-CD19-/CD56+ CD16+, and CD107a expression was evaluated.
10
Th17 and Treg Characterization in EAE
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