Purified splenic T cells from WT and G184S mice were labeled with 1 μM eFluor® 450, or 2.5 μM CMTMR for 15 min at 37°C and equal numbers of viable cells (8–10 million) were injected intravenously into recipient mice. After 2 h, spleen, inguinal lymph nodes, and popliteal lymph nodes were removed and gently dissociated into single cell suspensions. Peripheral blood was collected by retro-orbital eye bleeding. After removing red blood cells with Tris-NH4Cl, the cells were re-suspended in PBS containing 1% BSA at 4°C. LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (ThermoFisher) was used to exclude dead cells. Data acquisition was done on FACSCanto II flow cytometer and analyzed with FlowJo software (Tree Star). The egress assay used a similar initial approach, but two hours after the CD4+ T cell transfer mice were injected intravenously with CD62L antibody (100 μg/mouse). After 18hr spleen, inguinal lymph nodes, and popliteal lymph nodes were removed and gently dissociated into single cell suspensions. Peripheral blood was collected by retro-orbital eye bleeding. After removing red blood cells with Tris-NH4Cl, the cells were re-suspended in PBS containing 1% BSA at 4°C. The ratios between the numbers of cells in the various compartments two hours after transfer versus after CD62L antibody injection were calculated.
Facscanto 2 flow cytometer
Manufactured by Tree Star
Sourced in United States
The FACSCanto II flow cytometer is a versatile and powerful instrument designed for high-performance multiparameter analysis. It features a blue, red, and violet laser configuration, allowing for the detection and analysis of a wide range of fluorescent parameters. The FACSCanto II is capable of simultaneous measurement of up to 10 different fluorescent signals, making it a valuable tool for a variety of applications in cell biology and immunology research.
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Lab products found in correlation
Ionomycin, by Merck Group (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Live dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Pe annexin 5 apoptosis detection kit, by BD (1 mentions)
Annexin 5 pe 7 aad apoptosis assay kit, by BD (1 mentions)
Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Anti cd8 pe, by BD (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Cd49d, by BioLegend (1 mentions)
Fc block 2.4g2, by BD (1 mentions)
Igg1 fitc, by BD (1 mentions)
Igg2a pe, by BD (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Magnetic cell sorting system, by Miltenyi Biotec (1 mentions)
Fitc conjugated anti cd34, by BioLegend (1 mentions)
Normal rat serum, by Jackson ImmunoResearch (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Anti cd4 pe cy7, by Thermo Fisher Scientific (1 mentions)
20 protocols using facscanto 2 flow cytometer
1
Tracking T Cell Egress In Vivo
2
Apoptosis Detection by Flow Cytometry
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Apoptosis assays were performed using the PE Annexin V apoptosis detection kit (BD Biosciences) according to the manufacturer's instructions. Briefly, U87 and U251 cells were stained with Annexin V-PE and 7-AAD in the dark for 15 min at room temperature. Apoptotic cells (i.e., the total percentage of Annexin V-positive cells) were quantified using the FACS Canto II flow cytometer and FlowJo software (TreeStar, Ashland, OR, USA).
3
Tracking Splenic B Cell Migration
4
Measuring Apoptosis by Flow Cytometry
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Apoptosis was measured by flow cytometry using an annexin V-PE/7-AAD apoptosis assay kit (BD Pharmingen Biosciences, San Diego, CA, USA) according to the manufacturer's protocol.29 (link) Briefly, 2 × 105 cells were treated with SAHA for 24 h, incubated with annexin V-PE and 7-AAD for 15 min, and then analyzed by flow cytometry. Stained cells were analyzed using the FACSCantoII flow cytometer and the FlowJo software (Tree Star, Ashland, OR, USA).
5
CFSE-Based T Cell Proliferation Assay
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Cell proliferation was determined by the carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay using the CellTrace™ CFSE Cell Proliferation Kit (Invitrogen, USA) and was performed according to the manufacturer’s protocol. 2 × 106 PBMCs were stained with final concentration of 0.3 μM CFSE prior to culture. Then, 1.5 × 105 cells were cultured in a 96-wells plate in duplicate in the presence of PHA (positive control; Sigma-Aldrich Chemie GmbH, Germany), PPD and ESAT-6 (SSI) or left unstimulated (negative control) at 37 °C in a 5% CO2 humidified incubator. The final concentration of each antigens were 5 μg/ml. After 4 days (PHA) and 6 days (other conditions), the cells were harvested and stained with anti-CD3-APC, anti-CD4-PeCy5 and anti-CD8-PE (BD Biosciences). Cells were acquired by a FACSCanto II flow cytometer and data were analyzed by FlowJo (TreeStar, USA). Proliferating cells were defined as those with reduced (low or dim) CFSE expression and % specific proliferation defined as the fraction of cells with low CFSE after subtraction of unstimulated control cell values.
6
Comprehensive Tfh Cell Phenotyping by Flow
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For flow cytometry analyses of freshly isolated cells, a minimum of 350,000 PBMCs and 3,000 CSF cells were incubated with a FcR-blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with fluorochrome-conjugated antibodies against surface molecules of interest. For Tfh cell phenotyping, the following antigens were assessed: CD3 (AF488; UCHT1), CD127 (BV421; A01905), CD25 (PE; M-A251), PD-1 (BV605; EH12.2H7), CXCR5 (PE/Cy7; J252D4), CCR7 (AF674; G043H7), CD45RA (FITC; H100), CD69 (APC; FN50), CXCR3 (APC; G025H7), CCR2 (PerCP/Cy5.5; K036C2), CCR4 (APC; L291H4), CCR6 (PerCP/Cy5.5; G034E3), melanoma cell adhesion molecule 1 (BV421; P1H12), CD49d (APC; 9F10), SLAMF5 (FITC; MZ18-21F6) all from BioLegend (San Diego, CA, USA), HLA-DR (APC; L203) from R&D Systems (Minneapolis, MS), and CD4 (APC/AF750; S3.5) from Invitrogen (Waltham, MA). The corresponding isotype controls were included where appropriate. Data were acquired on a FACS Canto II flow cytometer, and data analysis was performed using FlowJo software (Tree Star, Inc., Ashland, OR).
7
Murine Subcutaneous Tumor Inoculation and Flow Cytometry
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2×106 UN-SCC679 or UN-SCC680 cells were subcutaneously inoculated in one flank of 6-8-week-old female A/J mice (Harlan-Winkelmann). Thirteen days after cell inoculation, tumors were collected and processed for flow cytometry analysis as previously described (Azpilikueta et al., 2016 (link)). Single-cell suspensions were treated with Fc block (2.4G2; BD Pharmingen) to avoid unspecific staining and then stained with the following fluorochrome-conjugated antibodies, as previously reported (Ajona et al., 2020 (link)). Data acquired in a FACSCanto II flow cytometer were analyzed using FlowJo software (Tree Star, Inc., Ashland, Oregon). The gating strategy used to analyze the data is shown in Fig. S2 .
8
Quantifying Monocyte and Macrophage Surface Markers
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CD14+ cells present in isolated PBMC were determined using anti-human CD14-PE (clone M5E2, BD Biosciences, Franklin Lakes, NJ) and an isotype matched negative control, IgG2a-PE (BD Biosciences). PBMC were stained for 30 minutes at 4°C, washed with 1% BSA in PBS and fixed with 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). Immunopositive cells were analyzed by acquisition of 10,000 events on a FACS Canto II flow cytometer and analyzed using FlowJo (Treestar, Ashland OR). To analyze CD4 or CCR5 protein expression on the macrophage surface, MDM were detached from culture dishes with TrypLE Select (10X) for 30 minutes at 37°C, followed by gentle agitation and scraping. 1–2×105 MDM were stained for CCR5 or CD4 using anti-human CCR5-APC/Cy7 and CD4-FITC, with the isotype-matched negative controls, IgG1-APC/Cy7 or IgG1-FITC (BD Biosciences). Antibodies were titrated to determine the optimal concentration for staining PBMC and MDM. After subtracting out the background fluorescence from the isotype matched control, the mean fluorescence intensity (MFI) for each antigen was determined.
9
Enrichment and Characterization of CD34+ Cells
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The cryopreserved BM samples were thawed and the CD34+ cells were enriched by positive selection using CD34 microbeads and a magnetic cell-sorting system (Miltenyi). Briefly, the BM cell were suspended in 100 μL of PBS/0.5% BSA/2 mM EDTA solution and were filtered through a 30 µm nylon mesh to remove cell clumps. Human FcR blocking reagent and anti-CD34 microbeads were added and then incubated at 4 °C for 30 min. Subsequently, cells were washed and resuspended in 500 μL PBS solution and were passed through the MS separation column kept in the magnetic field to hold the CD34+ cells in the column. Then, the purity of the isolated CD34+ cells was evaluated by flow cytometry; the post-sort purity was routinely >95% (Figure S1 ). In detail, 1 × 105 cells were suspended in 100 μL PBS and were stained with FITC-conjugated anti-CD34 (Biolegend) in the dark for 30 min on ice, and DAPI (1 μg/mL) was added to gated-out dead cells before running the samples. The samples were stained and analyzed on a FACS Canto II flow cytometer, and data was processed with FlowJo version 9 or 10 (Tree Star).
10
Isolation and Cytokine Analysis of Liver Leukocytes
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The liver leukocytes were recovered using Ficoll-Paque PLUS gradient centrifugation. After processing, the viability was assessed using Trypan blue exclusion and the cell concentration was determined. For cytokine staining, the cells were preincubated with 20 ng/ml of PMA, 500 ng/ml of ionomycin, and Golgi Plug for 6 h; permeabilized using a Cytofix/Cytoperm kit according to the manufacturer’s instructions; and stained with α-IL-17A conjugated with Alexa700 and α-IFN-γ conjugated with APC-CY7. For FoxP3 labeling, the Foxp3 Staining Kit was used according to the manufacturer’s recommendations. For each sample, data from a minimum of 200,000 cells were acquired using a FACSCanto II flow cytometer and analyzed using FlowJo software (Tree Star, OR, USA).
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