T cell subpopulations and the efficiency of T cell isolation from PBMCs were quantified by three-color staining using the following sets of mAbs: (1) anti-CD18 FITC (Becton–Dickinson, San Jose, CA, USA), anti-CD3 APC (eBioscience) and anti-CD4 PE (eBioscience) or (2) anti-CD18 FITC (BD), anti-CD3 APC (eBioscience) and anti-CD8 PE (eBioscience). Percentages of T cells (gated on CD18+ and CD3+) expressing CD4 or CD8 were measured by flow cytometry (Additional file 1 : Table S2, Figure S1). All FACS data were acquired on a FACS Verse (BD) using BD FACSuite™ software (BD). Data were analyzed using FlowJo software version 10 (Tree Star, Ashland, OR, USA).
Anti cd3 apc
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-CD3-APC is a fluorescently labeled antibody that recognizes the CD3 antigen, which is a component of the T cell receptor complex. It is commonly used in flow cytometry applications to identify and quantify T cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd4 fitc, by Thermo Fisher Scientific (8 mentions)
Anti cd8 pe, by Thermo Fisher Scientific (5 mentions)
Lsrii flow cytometer, by BD (4 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Anti cd4 pe, by Thermo Fisher Scientific (3 mentions)
Anti cd45 pe, by Thermo Fisher Scientific (3 mentions)
Anti cd3 pe, by BD (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Anti cd45 efluor450, by Thermo Fisher Scientific (2 mentions)
Anti cd19 pe, by BioLegend (2 mentions)
Anti cd4 fitc, by BioLegend (2 mentions)
Anti cd8 pe cy7, by BioLegend (2 mentions)
7 aad, by BioLegend (2 mentions)
Anti cd138 brillantviolet605, by BioLegend (2 mentions)
Collagenase d, by Merck Group (2 mentions)
Dnase, by Merck Group (2 mentions)
Anti flag fitc, by Merck Group (2 mentions)
Dapi fluoromount g, by Merck Group (2 mentions)
40 protocols using anti cd3 apc
1
Quantifying T Cell Subpopulations by Flow Cytometry
2
Flow cytometric analysis of bladder immune cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bladders were aseptically removed, minced with scissors, and digested at 37° for 30 minutes in RPMI-1640 with 10mM HEPES, collagenase D (C5318, Sigma-Aldrich), and DNAse (10104159001, Sigma-Aldrich). Bladders were forced through a 70 μm cell strainer (352350, Corning) and washed with 5% FBS in PBS. Single cell suspensions were stained with anti-CD45-eFluor450 (48–0451-82, eBioscience), anti-CD3-APC (17–0032-82, eBioscience), anti-CD19-PE (115511, BioLegend), anti-CD4-FITC (100405, BioLegend), anti-CD8-PE/Cy7 (100721, BioLegend), anti-CD138-BrillantViolet605 (142515, BioLegend), and 7-AAD (420404, BioLegend). Data was acquired on LSR II flow cytometer (BD) and analyzed with FlowJo software v10.0. Gates were determined with isotype antibodies in bladder suspensions from young mice.
3
Characterization of Th17 and Treg Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LPLs and pLN cells stimulated for 4 hours with PMA (50 ng/ml; Sigma), ionomycine (1 μg/ml; Sigma), and the Golgi-traffic inhibitor Brefeldin (1 μl/ml; BD Biosciences). Cells were stained with anti-CD3-PE (BD Pharmingen) or anti-CD3-APC (eBioscience) and anti-CD4-APC (Biolegend) or anti-CD4-FITC (BD Pharmingen). Next, the cells were fixed and permeabilized using fixation/permeabilization buffer (eBioscience). For intracellular staining the cells were incubated in permeabilization buffer (eBioscience) containing anti-IL-17-FITC (Biolegend), anti-IFNγ-FITC (BD Pharmingen), anti-IL-4-PE (BD Pharmingen) or Foxp3-FITC (eBioscience). An appropriate isotype matched control antibody was used in all FACS analyses. Cells were analyzed on a FACS Calibur using the CellQuest software (BD Biosciences). Results were analyzed with FlowJo version 7.6.5.
4
Germinal Center Formation in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To look at germinal center formation in mice, lungs and lymph nodes were harvested 24 days after the first challenge with NC99 H1N1. Lungs from each mouse were processed through 70 um cell strainer separately, red blood cells were lysed, and resuspended in FACS buffer. Collected lymph nodes from mice were pooled per group and made into single-cell homogenates by forcing them through a cell strainer. To stain the cells, Fc receptor were blocked using anti-CD16/CD32 (BD), anti-B220-AF700, anti-GL7-e420, anti-Fas-PE Cy7, anti-IgM-PerCpCy5.5, and anti-CD3-APC (all eBioscience) were used.
5
Th17 and Treg Cell Quantification
6
Multiparameter Analysis of Murine Tissues
7
Comprehensive Multiparametric Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell surface markers were stained with these specific antibodies: anti-CD19-PE, anti-CD3-APC, anti-CD4-PE, anti-CD8-FITC, 7-AAD, and anti-annexin-V-FITC (eBioscience); anti-CD19-APC, anti-B220-Per-cy5.5, anti-IgM-FITC, anti-AA4.1-PE, anti-CD23-eFluor647, and anti-CD8-APC (Biolegend).
For ex vivo analysis, cells were stimulated with 25 ng/mL PMA (Sigma-Aldrich) and 1 g/mL ionomycin (Sigma-Aldrich) in the presence of 0.66 μL/mL Golgistop (BD PharMingen) for 6 h at 37 °C, 5 % CO2. Intracellular staining of IFN-γ and IL-4 was performed using Transcription Factor Staining Buffer Set (eBioscience). Data was collected by FACS Calibur flow cytometer (BD Biosciences) and analyzed by FlowJo software (TreeStar, Ashland, OR).
For cell quantization, blood sample (100 μL) was stained with the specific antibodies: anti-CD19-FITC, anti-CD3-APC, anti-CD4-FITC, anti-CD8-FITC, anti-CD11c-APC, anti-F4⁄80-FITC, and anti-CD11b-APC. Erythrocytes were then lysed with Cal-lyse Lysing Solution (Invitrogen). After thoroughly mixing with 100 μL of Caltag Counting Beads (Invitrogen), 10,000 beads were acquired in the FACS Calibur flow cytometer for each sample.
For ex vivo analysis, cells were stimulated with 25 ng/mL PMA (Sigma-Aldrich) and 1 g/mL ionomycin (Sigma-Aldrich) in the presence of 0.66 μL/mL Golgistop (BD PharMingen) for 6 h at 37 °C, 5 % CO2. Intracellular staining of IFN-γ and IL-4 was performed using Transcription Factor Staining Buffer Set (eBioscience). Data was collected by FACS Calibur flow cytometer (BD Biosciences) and analyzed by FlowJo software (TreeStar, Ashland, OR).
For cell quantization, blood sample (100 μL) was stained with the specific antibodies: anti-CD19-FITC, anti-CD3-APC, anti-CD4-FITC, anti-CD8-FITC, anti-CD11c-APC, anti-F4⁄80-FITC, and anti-CD11b-APC. Erythrocytes were then lysed with Cal-lyse Lysing Solution (Invitrogen). After thoroughly mixing with 100 μL of Caltag Counting Beads (Invitrogen), 10,000 beads were acquired in the FACS Calibur flow cytometer for each sample.
8
PD-L1 Expression Analysis in Septic Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry was performed to detect the PD-L1 expression level in the PD-L1 humanized mice after CLP surgery. Blood samples were collected by heart puncture and anticoagulated in a BD Vacutainer (BD, Franklin Lakes, New Jersey, U.S.A.) 24 h after surgery. The white blood cells were stained with anti-CD3-APC, anti-CD11b-FITC, anti-Ly6C-APC, anti-Ly6G-APC, and anti-PD-L1-PE antibodies (purchased from eBioscience, San Jose, California, U.S.A.). Flow cytometry assays were performed with a FACSCalibur Flow Cytometer (BD Biosciences, Heidelberg, Germany) and the data were analyzed with FlowJo 7.6 software (Tree Star, Ashland, Oregon, U.S.A.). The level of PD-L1 expression was shown as the percentage of PD-L1-positive cells.
9
Detecting IL-17+ CD4+ T Cells in Spleen and Lung
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect IL-17+/CD4+ T cells among splenic cells, the cells were incubated with 10 μg/mL brefeldin A (eBioscience) for 2 h and then stained for cell surface CD4 using a FITC-conjugated anti-CD4 monoclonal antibody (mAb) (eBioscience) at 4°C for 30 min. After incubation in fixation/permeabilization solution (eBioscience), the cells were stained for intracellular IL-17 using a PE-conjugated anti-IL-17 mAb (eBioscience) for 30 min and then analyzed using a FACSCalibur flow cytometer (BD Biosciences).
To detect IL-17+/CD4+ T cells in the lung tissue, lung cells were obtained according to a previously reported method [15 (link)]. Lung cells (4 × 106/mL) were washed three times in FACS buffer (PBS containing 1% bovine serum albumin and 0.1% sodium azide), incubated with brefeldin A (10 μg/mL) for 2 h, and then stained with surface-specific mAbs (anti-CD3-APC and anti-CD4-FITC; eBioscience) for 30 min at 4°C. For intracellular staining, cells were fixed and permeabilized in fixation/permeabilization solution, intracellularly stained with the PE-conjugated anti-IL-17 mAb for 30 min, and then analyzed using the FACSCalibur flow cytometer.
To detect IL-17+/CD4+ T cells in the lung tissue, lung cells were obtained according to a previously reported method [15 (link)]. Lung cells (4 × 106/mL) were washed three times in FACS buffer (PBS containing 1% bovine serum albumin and 0.1% sodium azide), incubated with brefeldin A (10 μg/mL) for 2 h, and then stained with surface-specific mAbs (anti-CD3-APC and anti-CD4-FITC; eBioscience) for 30 min at 4°C. For intracellular staining, cells were fixed and permeabilized in fixation/permeabilization solution, intracellularly stained with the PE-conjugated anti-IL-17 mAb for 30 min, and then analyzed using the FACSCalibur flow cytometer.
10
Quantitative Analysis of Immune Cells in BAL
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BAL were obtained and analyzed one day after the last challenge (7 to 9 mice per group). One mL of PBS was instilled intra-tracheally until lungs are inflated uniformly and then slowly aspirated and centrifugated. Total cell number was determined on Kova slide® by optical microscopy. Differential cell counts are determined by flow cytometry with flow cytometer BD LSR II after staining with anti-CD3 APC, anti-CD19 PE-Cy7, FITC anti-F4/80 (eBiosciences, paris, France), anti-CCR3 PE (R&D, Lille, France), anti-PerCP-Cy5.5 Ly6G, anti-CD8 APC-H7 (BD Biosciences, Le Pont-de-Claix,France) and DAPI. Leukocyte populations were identified as neutrophils (Ly6G high), macrophages (F4/80 high DAPI mid (autofluorescence)), eosinophils (CCR3 high), B cells (CD3 négative CD19 high), T cells (CD3 high CD19 négative), and the proportion of CD8+ T cells is determined by the CD8 high population.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!