For flow cytometric analysis, isolated intestinal ECs were stained with UEA-1-TRITC, anti-CD45–Pacific blue (PB; Biolegend, San Diego, CA), and Viaprobe (BD Biosciences, East Rutherford, NJ). Viaprobe− CD45− UEA-1+ cells were identified as F-ECs. After blocking with anti-CD16/32 (FcγRII/III) (BD Biosciences), the following antibodies were used to stain spleen and LP cells: anti-CD45–PB (Biolegend), anti-CD11b–phycoerythrin (PE), anti-Foxp3-fluorescein isothiocyanate (FITC) (eBioscience, San Diego, CA), anti-CD11c–allophycocyanin (APC), anti-CD11b–FITC, anti-Gr-1-Alexa647, anti-CD3-APC, anti-B220-PE, anti-B220-APC, anti-IgA-FITC, anti-CD4-eFluor450, anti-CD90.2–FITC, anti-IL-17-PE, and anti-IFNγ-FITC (all from BD Biosciences), and Viaprobe. CD11b− CD11c− CD19− LP cells were purified by using anti-CD11b, anti-CD11c, and anti-CD19 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The results were obtained by using a FACSAria cell sorter (BD Biosciences) with FlowJo software (TreeStar, Ashland, Oregon).
Via probe
Manufactured by BD
Sourced in United States, France
The Via-Probe is a compact and versatile laboratory instrument designed for precise measurement and monitoring. It functions as a multi-purpose data acquisition device, capable of recording and transmitting various types of sensor data in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Magnetic column, by Miltenyi Biotec (5 mentions)
Anti pe magnetic beads, by Miltenyi Biotec (5 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Facscanto 2, by BD (4 mentions)
Software, by FlowJo (3 mentions)
Cd45ra, by Thermo Fisher Scientific (3 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (2 mentions)
Lsr fortessa cytometer, by BD (2 mentions)
Cmac blue assay, by Thermo Fisher Scientific (2 mentions)
Collagenase cls1, by Worthington (2 mentions)
Zombie uv fixable viability kit, by BioLegend (2 mentions)
Facsaria 2, by BD (2 mentions)
Cd4 apc clone rpa t4, by Thermo Fisher Scientific (2 mentions)
Cd14 percp clone mφp9, by BD (2 mentions)
Cd45ro fitc clone uchc1, by Thermo Fisher Scientific (2 mentions)
Cd19 percp clone sj25c1, by BD (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Pe magnetic beads, by Miltenyi Biotec (2 mentions)
Facsmelodytm cell sorter, by BD (2 mentions)
Percoll, by Merck Group (2 mentions)
47 protocols using via probe
1
Flow Cytometric Analysis of Intestinal Immune Cells
2
Quantifying Antigen-Specific T-Cell Frequencies
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Analysis of T-cell frequency was accomplished using our previously published approach (26 (link)). Briefly, 40–60 × 106 PBMCs were resuspended in a total of 400–600 µL media, divided into two or three independent tubes of 20 × 106 cells (200 µL) each, incubated with 50 nmol/L dasatinib for 10 min at 37°C, and stained with 20 µg/mL PE-labeled, PE-CF594–labeled, or PE-Cy5–labeled HIP tetramers at room temperature for 120 min (three tetramers per tube for a total of six hybrid peptide tetramers plus an nonimmunogenic control tetramer if adequate cells were available for a third staining tube). Cells were washed, incubated with PE-magnetic beads (Miltenyi Biotec) for 20 min at 4°C, and magnetically enriched; 1% of the cells were retained as a nonenriched sample. Enriched (bound) and nonenriched (precolumn) samples were stained with CD4 V500, CD14 PerCP-Cy5.5, and CD19 PerCP-Cy5.5 (eBioscience) and CD45RA AF700 (BD), CXCR3 FITC, CCR6 BV421, and CCR4 BV605 (BioLegend) for 15 min at 4°C. After washing, cells were labeled with ViaProbe (BD Biosciences) and analyzed on a FACSCanto (BD Biosciences), gating on CD4+CD14−CD19−ViaProbe− cells and plotting tetramer versus CD45RA. Frequencies were calculated as previously described (26 (link)).
3
Isolation of Intestinal Epithelial Cells
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The protocol for isolation of IEC cells was based on the previously described method with minor modifications. In brief, the small and large intestines were harvested individually from treated mice and rinsed extensively with RPMI-1640 media (from Lonza) after Peyer’s patches were removed (for small intestine). The rinsed intestines were opened longitudinally and macerated; the tissue pieces were shaken gently in RPMI-1640 containing 2 mM EDTA and 10% fetal calf serum. The tissue preparations were passed through 70-µm mesh filters, and the resulting single-cell suspensions were applied to Percoll (from Sigma) density gradients of 25%, 40%, and 75%. After centrifugation at 2000×g for 20 min, the interface between the 25% and 40% layers was collected to obtain IECs. The cells were stained using antibodies for either epithelial cell adhesion molecule (EpCAM, from Biolegend) or CD45 (from Biolegend) and nucleic acid dye (Via-Probe, from BD Biosiences). The Via-Probe−/CD45−/EpCAM+ IEC were sorted using BD FACSMelodyTM Cell Sorter (BD Biosciences) for further biomedical analysis [55 (link), 56 (link)].
4
Isolation and Sorting of Intestinal Epithelial Cells
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The protocol for isolation of IECs was based on the previously described method with minor modifications. In brief, the small and large intestines were harvested individually from treated mice and rinsed extensively with RPMI‐1640 media (from Lonza) after Peyer's patches were removed (for small intestine). The rinsed intestines were opened longitudinally and macerated; the tissue pieces were shaken gently in RPMI‐1640 containing 2 mM EDTA and 10% fetal calf serum. The tissue preparations were passed through 70‐μm mesh filters, and the resulting single‐cell suspensions were applied to Percoll (from Sigma) density gradients of 25%, 40%, and 75%. After centrifugation at 2000× g for 20 min, the interface between the 25% and 40% layers was collected to obtain IECs. The cells were stained using antibodies for either epithelial cell adhesion molecule (EpCAM, from Biolegend) or CD45 (from Biolegend) and nucleic acid dye (Via‐Probe, from BD Biosciences). The Via‐Probe– CD45– EpCAM+ IECs were sorted using the BD FACSMelodyTM Cell Sorter (BD Biosciences) for further biological assays.36 , 37
5
Enrichment Protocol for T-cell Frequency Analysis
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Direct analysis of T-cell frequency was accomplished using our previously published enrichment approach (12 (link)). Briefly, 30–50 × 106 PBMCs were resuspended in 200 µL of T-cell media, incubated with 50 nmol/L dasatinib for 10 min at 37°C, and stained with 20 μg/mL phycoerythrin (PE)-labeled tetramer at room temperature for 100 min, followed by antibody staining with CD4-APC (eBioscience), CD45RO-FITC (eBioscience), and a combination of CD14-PerCP and CD19-PerCP (BD Biosciences) for 15 min at 4°C. Cells were washed, incubated with PE magnetic beads (Miltenyi Biotec) for 20 min at 4°C, and enriched with a magnetic column, retaining 1% of the cells as a nonenriched sample. The PE-enriched and precolumn samples were labeled with Via-Probe (BD Biosciences) and analyzed on a FACSCalibur (BD Biosciences), gating on CD4+CD14−CD19−Via-Probe− and plotting tetramer versus CD45RO. Frequencies were calculated as previously described (12 (link)). Statistical analysis was performed using unpaired t tests with Welch correction with Prism software (version 5.03, GraphPad Software Inc.).
6
FACS Isolation of OSR1-GFP+ Cells
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Cells were detached using Accumax® (Innovative Cell Technologies, San Diego, CA), collected, and then washed with Hank’s Balanced Salt Solution (Thermo Fisher Scientific). Dead cells ware stained with BD Via-Probe™ (BD Biosciences, San Jose, CA). Cells were sorted using a fluorescence-activated cell sorter (FACS) Aria II (BD Biosciences), according to the manufacturer’s instructions. OSR1-GFP+ cells were collected in Stage 2 medium containing 10 μM Y-27632 (Wako, Tokyo, Japan).
7
Bleomycin-induced Mitochondrial Stress Assay
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Overnight yeast cells grown in 3 mL liquid SD medium at 30°C with constant agitation were collected at 3,000 × g for 2 min the next morning and transferred in SD and SD+Etn medium for 2 to 4 h. The initial cell density was adjusted to around OD600 = 0.3 before treatment with bleomycin (2.5 μM for Sc and 5 μM for H99) for 10 h. Cells were harvested, washed once in PBS, and resuspended in PBS supplemented with 0.2 μM MitoTracker Deep Red FM (Invitrogen) and 1 μg of Hoechst 33342 (Thermo Fisher Scientific). Cells were incubated for 10 min at 30°C in the dark, washed twice with PBS, resuspended in PBS, and observed under a fluorescence microscope (Olympus BX61, Olympus Life Sciences). MitoTracker Deep Red fluorescence was detected by flow cytometry with a BD Via-Probe (BD Biosciences) using FL3 channel. The data were analyzed using FlowJo software (FlowJo LLC) and expressed as mean fluorescence intensity (MFI). All experiments were done in triplicate. Analysis was performed using the unpaired, two-tailed t test in GraphPad Prism version 8. P values of <0.05 were considered statistically significant.
8
Cord Blood Leukocyte Phenotyping
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EDTA-treated cord blood samples were analyzed by flow cytometry (Beckman Coulter Navios flow cytometer, Kaluza 1.2 software). Leukocytes were gated and differentiated from debris according to their forward and side scatter. BD Via-Probe (BD Biosciences, San Jose, CA) cell viability solution (7-AAD labeled) was used to differentiate living from dead cells. Cell surface CD45 (label: FITC, isotype: IgG1, κ, Clone: HI30) and CD34 (label: PE-Cy7, isotype: IgG1, κ, Clone: 581) were stained with specific fluorescent antibodies (BioLegend, San Diego, CA). Residual red blood cells (RBC) were filtered out from the leukocytes according to their absent CD45 expression.
9
Granulocyte Cell Death Assay
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Blood samples were taken from the sub-orbital sinus of three CDF1 mice directly into dry EDTA tubes. Each blood sample was divided and used for all four groups included in the study: 1) CA4P, 2) CA4, 3) DMSO, and 4) saline. To 100 µl whole blood 2 µl of CA4P, CA4, or saline were added (the final concentrations of CA4P and CA4 were 10 µM). Because a single drop of DMSO is required to dissolve CA4 we included group 3, where 2 µl of a solution containing one drop of DMSO in 5 ml saline was added to 100 µl whole blood. All samples were incubated for two hours at 37°C. The numbers of dead and/or apoptotic granulocytes in each group were determined using flow cytometry as described above using Annexin V (BD Pharmingen, Broendby, Denmark) and BD Via-Probe (BD Biosciences).
10
Immune Response to Streptococcus gallolyticus
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Inoculation of whole blood with S. gallolyticus subsp. gallolyticus strains was performed with human blood from three healthy volunteers. Blood was collected in heparin-gel monovettes (Kabe, Nümbrecht-Elsenroth, Germany) and mixed with RPMI1640 with or without S. gallolyticus subsp. gallolyticus from overnight cultures (initial bacterial titer: 1.6–4.8 × 105 colony-forming units (CFU)/ml; volume ratio 1:10), as described previously [25 (link)]. Suspensions were incubated in 6-well plates at 37 °C and 5% CO2. Aliquots of 500 μl samples from either bacterial, blood or medium suspensions were used for the plating assay after 0, 6, 24 and 48 h. The experiment was performed on two different days with three technical replicates/day/volunteer. The residual probes were centrifuged at 1000 × g for 5 min and the supernatant was used for the measurement of IL-6 with a human IL-6 enzyme-linked immunosorbent assay (ELISA; Thermo Scientific, Waltham, USA), following the manufacturer’s instructions. Cell viability of leukocytes in whole blood was determined by flow cytometry (FACSCanto II; BD Biosciences, San José, USA) after lysis of the erythrocytes with BD Viaprobe (1:200).
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