Thymuses were collected from OT-I Rag2-/-, F5 Rag1-/-, or B6 mice and dissociated through a 70 μm cell strainer to yield a cell suspension. Thymocytes were then labeled with 1 μM Cell Proliferation Dye eFluor450 or 0.5 μM Cell Proliferation Dye eFluor670 (Thermo Fisher Scientific) at 107 cells/ml at 37°C for 15 min in PBS, then washed and resuspended in complete RPMI (containing 10% FBS, penicillin streptomycin, and 2-mercaptoethanol, cRPMI) for overlay onto thymic slices. Thymocytes do not proliferate at the timepoints collected, allowing overlaid thymocytes to be distinguished from slice resident thymocytes by Cell Proliferation Dyes (Figure 1a ). In imaging experiments, OT-I thymocytes were depleted of mature CD8 single positives using the EasySep Biotin Positive Selection Kit (Stemcell Technologies) with anti-human/mouse β7 integrin antibody (FIB504, Biolegend) according to the manufacturer’s instructions. Thymocytes were then labeled with 3 μM SNARF (Thermo Fisher Scientific) at 107 cells/ml at 37°C for 15 min in PBS, then washed and labeled with 5 μM Hoechst 33342 (Thermo Fisher Scientific) at 107 cells/ml at 37°C for 15 min. For all other experiments, the total, unfractionated thymocyte population was used.
Snarf
Manufactured by Thermo Fisher Scientific
Sourced in United States
SNARF is a fluorescent pH indicator that can be used to measure intracellular pH in live cells. It exhibits a shift in its emission spectrum in response to changes in pH, allowing for ratiometric measurements of pH levels.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Cell proliferation dye efluor 450, by Thermo Fisher Scientific (2 mentions)
Cell proliferation dye efluor 670, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Easysep biotin positive selection kit, by STEMCELL (1 mentions)
Anti il 4 11b11, by BioXCell (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Anti cd4 percp cy5, by BD (1 mentions)
Anti ifn γ fitc, by BD (1 mentions)
Anti par3, by Merck Group (1 mentions)
Anti cd28 clone cd28, by BD (1 mentions)
Live dead fixable near ir, by Thermo Fisher Scientific (1 mentions)
Cell tracker violet, by Thermo Fisher Scientific (1 mentions)
Anti γ tubulin gtu 88, by Merck Group (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Anti human cd3ε okt3, by American Type Culture Collection (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Maxima sybr green master mix, by Thermo Fisher Scientific (1 mentions)
9 protocols using snarf
1
Thymocyte Labeling and Imaging
2
Antibody-based Characterization of T Cell Activation
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Antibodies used were anti-human CD3ε (OKT3, American Type Culture collection, Manassas, VA), anti-CD28 (clone CD28.2, BD Biosciences), and anti-TSAd-DyLight 488 (clone OTI3C7, Origene), anti–IL-4 (11B11, Bio X Cell), anti-IFNγ-FITC, anti-CD4-PerCP-Cy5.5 (RM4–5) and anti-CD44-FITC (KM201, BD), anti-CD62L-PE (MEL-14, Southern Biotech), anti-γ–tubulin (GTU-88, Sigma) and anti- Id-specific TCR-PE (GB11354 (link)). Polyclonal antibodies were anti-PAR3 (Cat# 07-330, Millipore), anti-PKCξ (Η - 1), anti-PKCθ (C-18), anti-Scrib (C-6) and anti-SAP97 (H-60, Santa Cruz). Secondary antibodies were goat anti-donkey, goat anti-rabbit or isotype specific anti-mouse conjugated to Alexa Fluor 488 (Thermo Fisher Scientific). Cytkines were IL-2, IL-12 (Peprotech). Fluorescent markers were phalloidin Alexa Fluor 647, cell tracker violet (CTV), SNARF, LIVE/DEAD Fixable Near-IR (all from Thermo Fisher).
3
Aorta-T Cell Interaction Assay
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Isolation of aortas and T cells were performed as previously described9 (link). Briefly, CD4+ T cells were purified from spleen and paLN using Robosep negative selection kit (StemCell Technology) and labeled for 10 min at 37 °C with 2.5 µM SNARF (Molecular Probes). Aortas were surgically removed from Apoe−/− or Apoe−/−Il27ra−/− mice fed with WD for 16 weeks and incubated with 5 × 105 CD4+ T cells obtained from spleens and paLNs of Apoe−/−Il27ra−/− mice fed WD for 16 weeks. T cells were incubated with the explanted aorta for 12 hours in 750 μl of complete RPMI 1640 media without any additional stimulation. In preparation of live image acquisition, the ends of each aorta were glued to a coverslip with Histoacryl glue (TissueSeal LLC), put in a Petri dish, maintained at 37 °C and superfused with RPMI medium 1640 without phenol red (Invitrogen) bubbled with a gas mixture containing 95% O2 and 5% CO2.
4
In Vitro T Cell Cytotoxicity Assay
5
Thymocyte Calcium Signaling Dynamics
6
PC12 Cell Line Characterization
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All reagents, if not separately mentioned, were purchased from Sigma-Aldrich (Germany). The PC12 rat pheochromocytoma cell line was obtained from ATCC (USA) and from Sigma-Aldrich (Germany). RPMI 1640 medium was from PAA (Austria). Calf and horse sera were from BioChrom (UK). Maxima SYBR Green Master Mix was from Fermentas (Canada). M-MLV Reverse Transcriptase, Trizol, Alexa Fluor 488, MitoTracker Red 580, MitoTracker Green TM, Fura-2 AM and SNARF were from Life Technologies (USA). Total RNA isolation kit was from Epicentre Biotech. (USA). Protein Assay Kit was from Bio-Rad (USA). TurboFect transfection regent was from Thermo Scientific. Primary antibodies against GFP, GAPDH, PMCA2 and PMCA3 were from Santa Cruz Biotech. (USA). Paq5000 polymerase was from Stratagene (USA). Phosphothioate oligodeoxynucleotides were from IDT (USA). Primers were synthesized in Institute of Biochemistry and Biophysics (Poland). mitoSypHer construct was kindly donated by Dr. Nicolas Demaurex.
7
Balanced Salt Solution Buffer Preparation
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Balanced salt solution (BSS) buffer contained 156 mM NaCl, 3.0 mM KCl, 1.25mM KH2PO4, 2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, 10 mM Hepes at pH 7.4. The KH2PO4 was removed in experiments employing Zn2+ which is precipitated by phosphate.
SNARF was from Invitrogen. Inhibitors were from either Sigma or Tocris and solubilized in dimethyl sulphoxide (DMSO) or 100% ethanol.
SNARF was from Invitrogen. Inhibitors were from either Sigma or Tocris and solubilized in dimethyl sulphoxide (DMSO) or 100% ethanol.
8
Adoptive Transfer and Intravital Imaging of OT-I CD8+ T Cells
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OT-I CD8+ T cells were isolated from the lymph nodes of UBC-GFP Rag1−/− OT-I TCR transgenic mice, and 5 × 106 cells were adoptively transferred into B6 recipient mice by i.v. injection. 18 h later, the OVA257–264 peptide SIINFEKL (OVAp) was injected i.v. (50 µg). At different times after peptide injection (3, 24, 30, 48, and 72) lymph nodes were harvested and digested in RPMI with 1 mg/ml collagenase and 0.1 mg/ml DNase (Sigma) for 20 min at 37°C. Alternatively, mice were anesthetized and the popliteal lymph nodes prepared for intravital imaging as previously described (Moreau and Bousso, 2017 (link)). In some experiments, a second cohort of OT-I CD8+ T cells labeled with 2.5 µM SNARF (Invitrogen) was adoptively transferred into mice by i.v. injection 18 h before intravital imaging.
9
Evaluating Cellular Oxidative Stress and Viability
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In addition to the life cell imaging, a portion of the cells was prepared for flow cytometry (FACSCalibur, Becton & Dickinson, Heidelberg, Germany) to analyze the ROS production with a second method and to compare the vitality among the four groups using CellQuest Pro software (version 5.2, Becton-Dickinson Bioscience, San Jose, USA) and FlowJo (version 10.0.7, FlowJo LLC, Ashland, OR, USA). In terms of cell vitality, propidium iodide- (PI-) negative cells were considered vital.
The materials and methods for quantifying ROS production have been described in detail in previous papers [22 (link), 23 (link)]. Briefly, cells were preincubated in 500 μL of HEPES buffer and Tyrode solution (modified to 1 mM CaCl2 and 1 mM MgCl2), 5 μL of DHR (10 μM), and 5 μL of seminaphtharhodafluor (SNARF, 10 μM, Invitrogen). Stimulation was induced by adding either 5 μL of fMLP (10 μM) and 5 μL of human tumor necrosis factor alpha (TNFα, 1 μg/mL, PeproTech Inc., Rocky Hill, NJ, USA) or 5 μL of phorbol-12-myristate-13-acetate (PMA, 10 μM, Sigma-Aldrich GmbH). Finally, 5 μL of PI (33671, 1.5 mM, Serva Electrophoresis GmbH, Heidelberg, Germany) was added to detect dead cells.
The materials and methods for quantifying ROS production have been described in detail in previous papers [22 (link), 23 (link)]. Briefly, cells were preincubated in 500 μL of HEPES buffer and Tyrode solution (modified to 1 mM CaCl2 and 1 mM MgCl2), 5 μL of DHR (10 μM), and 5 μL of seminaphtharhodafluor (SNARF, 10 μM, Invitrogen). Stimulation was induced by adding either 5 μL of fMLP (10 μM) and 5 μL of human tumor necrosis factor alpha (TNFα, 1 μg/mL, PeproTech Inc., Rocky Hill, NJ, USA) or 5 μL of phorbol-12-myristate-13-acetate (PMA, 10 μM, Sigma-Aldrich GmbH). Finally, 5 μL of PI (33671, 1.5 mM, Serva Electrophoresis GmbH, Heidelberg, Germany) was added to detect dead cells.
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