The cells were detached with TrypLE™ Express and washed with PBS once before use. The cells were suspended in 0.1 µM calcein AM diluted in DMEM without phenol red at a cell concentration of 1 × 106 cells/mL. The cells were incubated with calcein AM at 37 °C for 30 min, protected from light, and cells in suspension were intermittently agitated. The cells were kept on ice until the flow cytometry measurements. Cellular fluorescence reflecting the transport activity was measured by an Attune Nxt cytometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a blue (488 nm) laser. The calcein signal was detected in the BL1 channel (emission filter: 530/30 nm). Analysis of the data was carried out by the Attune Nxt cytometer Software v3.1.2 (Thermo Fisher).
Attune nxt cytometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Attune NxT cytometer is a flow cytometry instrument designed for multicolor analysis and cell sorting. It uses advanced optics, fluidics, and detection technologies to enable high-performance data acquisition and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cell stimulation cocktail, by Thermo Fisher Scientific (4 mentions)
7 aad, by BioLegend (3 mentions)
Attune nxt no wash no lyse filter kit, by Thermo Fisher Scientific (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Rnase a, by Merck Group (2 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Cd16 cd32 fcr blockers, by BioLegend (2 mentions)
High throughput autosampler, by Thermo Fisher Scientific (2 mentions)
Hm40 3, by BD (2 mentions)
Cd56 pe, by BioLegend (2 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
Red blood cell lysis buffer, by BioLegend (2 mentions)
Attune nxt software, by Thermo Fisher Scientific (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Sigmaplot 13, by Grafiti LLC (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (2 mentions)
Anti gr1 pe clone rb6 8c5, by BD (1 mentions)
Fitc anti ly 6c clone al 21, by BD (1 mentions)
Percp cy5.5 anti cd11b clone m1 70, by BD (1 mentions)
91 protocols using attune nxt cytometer
1
Quantifying Cell Membrane Transport Dynamics
2
Treg Cell and MDSC Quantification
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The CD4+Foxp3+ (Treg cells) and MDSC were measured by flow cytometry at day 7 post immunization protocol and at day 21 post infection. Briefly, mice were sacrificed and spleen single cell suspensions were prepared by homogenization through a stainless steel mesh using PBS 3% fetal bovine serum (FBS). Spleen absolute numbers were determined by counting cells in Neubauer chamber using Turk solution and then red blood cells were eliminated by lysis with H20 during 30 seconds. Finally, spleen cells were homogenized in PBS 3% FBS and 1.106 cells were stained with the following antibodies from BD-Pharmingen: fluorescein isothiocyanate (FITC) anti-CD4 (clone RM4-5), FITC anti-Ly6C (clone AL-21), phycoeritrin (PE) anti-Ly6G (clone 1A8), PE anti-GR1 (clone RB6-8C5) and PerCP-Cy 5.5 anti-CD11b (clone M1/70). Cells marked with CD4 were also intracellular stained for PE anti-Foxp3, using mAb from Miltenyi Biotec, according to manufacturer´s instructions.
Samples were acquired on an Attune NxT cytometer (Invitrogen) and analyses were performed using FlowJo software.
Samples were acquired on an Attune NxT cytometer (Invitrogen) and analyses were performed using FlowJo software.
3
Single-cell flow cytometry analysis
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Single-cell suspensions were stained with antibodies against appropriate extracellular markers (Supplementary Table 1 Supplementary Figure 2
4
Quantifying Mitochondrial H2O2 Accumulation
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For quantification (%) of GMCs with H2O2 accumulated specifically in mitochondria we used MitoPY1 (FITC+; #SML0734—Sigma-Aldrich, St. Louis, MO, USA) by flow cytometry (Attune NxT cytometer, Invitrogen; FlowJo software, BD Biosciences). All experiments were done three times in triplicate and the mean of each experiment was calculated.
5
Flow Cytometric Analysis of Coated Vaccinia Virus
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To determine whether coating of VV was successful, flow virometry of VV coated with Chol-PEG(10K)-FITC (named FITC-PCVV) was performed.
YOYO-3 (Thermo Fisher Scientific, Y3606) was used for nucleic acid staining of VV.13 (link),44 VV was incubated with 1 μM YOYO-3 at room temperature for 20 min. The sample was then centrifuged at 20,000 × g for 10 min. The viral pellet was washed three times in PBS as described in section 4.1. Final virus pellets were either resuspended in PBS to a concentration of 5.3 × 105 virus particles (VG)/μL and further modified to form PCVV, or 2.6 × 105 VG/μL as the naked form.44 Stained VV virions further modified to PCVV were coated and purified as detailed in section 4.1, and resuspended at a final concentration of 2.6 × 105 VG/μL. An Attune NxT cytometer (Invitrogen) was adapted to record FSC and SSC through the violet channel using the Attune NxT No-Wash No-Lyse filter kit (Thermo Fisher Scientific, 100022776). VV populations were gated for positive YOYO-3 staining. Analysis was performed using FlowJo v10.6.1 software. FITC-PCVV-YOYO-3 and VV-YOYO-3 were gated for positive YOYO-3 and FITC staining.
YOYO-3 (Thermo Fisher Scientific, Y3606) was used for nucleic acid staining of VV.13 (link),44 VV was incubated with 1 μM YOYO-3 at room temperature for 20 min. The sample was then centrifuged at 20,000 × g for 10 min. The viral pellet was washed three times in PBS as described in section 4.1. Final virus pellets were either resuspended in PBS to a concentration of 5.3 × 105 virus particles (VG)/μL and further modified to form PCVV, or 2.6 × 105 VG/μL as the naked form.44 Stained VV virions further modified to PCVV were coated and purified as detailed in section 4.1, and resuspended at a final concentration of 2.6 × 105 VG/μL. An Attune NxT cytometer (Invitrogen) was adapted to record FSC and SSC through the violet channel using the Attune NxT No-Wash No-Lyse filter kit (Thermo Fisher Scientific, 100022776). VV populations were gated for positive YOYO-3 staining. Analysis was performed using FlowJo v10.6.1 software. FITC-PCVV-YOYO-3 and VV-YOYO-3 were gated for positive YOYO-3 and FITC staining.
6
Flow Cytometry Analysis of Cell Death
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Cell death induction in GMCs was evaluated by Annexin-V (FITC+; #V13241—Thermo Fisher Scientific, Waltham, MA, USA), propidium iodide (PE+; #V13241—Thermo Fisher Scientific, Waltham, MA, USA) and DAPI (Violet+; #62248—Thermo Fisher Scientific, Waltham, MA, USA) staining by flow cytometry (Attune NxT cytometer, Invitrogen; FlowJo software, BD Biosciences). All experiments were done three times in triplicate and the mean of each experiment was calculated.
7
Mitochondrial Polarization in GMCs
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Polarized/depolarized mitochondria ratio in GMCs was evaluated by MitoProbe JC1 (#M34152—Thermo Fisher Scientific, Waltham, MA, USA) by flow cytometry (Attune NxT cytometer, Invitrogen and FlowJo software, BD Biosciences). Functional (polarized) mitochondria attract and aggregate JC1 probe, which shifts fluorescence emission from 530 nm (diffused probe—FITC+) to 590 nm (oligomerized probe—PE+). Results were expressed as PE+/FITC+ ratio. All experiments were done three times in triplicate and the mean of each experiment was calculated.
8
Caspase-3/7 Activity in β-cells
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INS-1 832/13 β-cells were trypsinized from the growth plate and stained per the manufacturer’s recommendation using a CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit to test Caspase-3/7 activity (Invitrogen, Cat. No. C10427) [26 (link)]. After staining, data were acquired using an Attune NxT cytometer (Invitrogen). Data were analyzed using FlowJo (BD Life Sciences, Franklin Lakes, NJ, USA, version 10.8.1).
9
Flow Virometry of aMUC1-PEG-Chol Labeled VV
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The samples were stained for aMUC1 using a goat anti-mouse AF647 secondary antibody (Invitrogen, A-21235). VV populations were determined using a YOYO-3 nucleic acid stain (Thermo Fisher Scientific, Y3606) and an anti-VV antibody (Abcam, ab35219). Preparation and washing were performed as described in section 4.1, with VV incubated for 1 h in excess aMUC1-PEG-Chol (10 mg/mL) solutions and washed vigorously. Flow virometry was performed at a final concentration of 6.35 × 104 VG/μL. An Attune NxT cytometer (Invitrogen) was adapted to record FSC and SSC through the violet channel using the Attune NxT No-Wash No-Lyse filter kit (Thermo Fisher Scientific, 100022776). Analysis was performed using FlowJo v10.6.1 software.
10
Assessing Cell Viability and Apoptosis
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For viability measurement, cells were plated at 100,000 cells/mL in medium containing DMSO or 0.5 mM OSU-13 and incubated for 24, 48, and 72 hours (h) at 37°C and 5% CO2. Afterwards, cells were stained with Zombie-aqua (Life Technologies; Carlsbad, CA, USA) according to manufacturer’s instructions. Cells were then analyzed using an Attune Nxt cytometer (Invitrogen; Waltham, MA, USA), and FlowJo software (FlowJo LLC; Ashland, OR, USA).
For apoptosis and necroptosis inhibition assays, OPM-2 cells (250,000 cells/mL) were pre-incubated for 1 h at 37oC in medium containing 100 mM of the general caspase inhibitor Z-VAD-FMK (Sigma-Aldrich; St Louis, MO, USA) or the necroptosis inhibitor necrostatin-1s (Cell Signaling Inc.; Danvers, MA, USA). Then, cells were washed and incubated for 72 h in medium containing DMSO or 1 mM OSU-13, stained with Zombie-aqua (Life Technologies), and cell viability was assessed as previously described.
For apoptosis and necroptosis inhibition assays, OPM-2 cells (250,000 cells/mL) were pre-incubated for 1 h at 37oC in medium containing 100 mM of the general caspase inhibitor Z-VAD-FMK (Sigma-Aldrich; St Louis, MO, USA) or the necroptosis inhibitor necrostatin-1s (Cell Signaling Inc.; Danvers, MA, USA). Then, cells were washed and incubated for 72 h in medium containing DMSO or 1 mM OSU-13, stained with Zombie-aqua (Life Technologies), and cell viability was assessed as previously described.
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