Phenotypes and activation markers were evaluated by Miltenyi Biotec flow cytometer-MACSQuant Analyzer (8 fluorescence channels, three lasers) on freshly isolated peripheral blood mononuclear cells. Immune activation was evaluated by multiparameter flow cytofluorimetric analysis by the following anti-human monoclonal antibodies: CD3-PerCP, CD4-APC-Vio770, CD8-FITC, CD45RO-PE-Vio770, CD27-VioBlue, CD38-APC, and HLA-DR-PE (Miltenyi Biotec, Bergisch Gladbach, Germany).
Cd8 fitc
Manufactured by Miltenyi Biotec
Sourced in Germany
CD8-FITC is a fluorescently labeled antibody that binds to the CD8 surface marker, which is commonly expressed on a subset of T cells. It can be used for the identification and analysis of CD8+ T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 apc vio770, by Miltenyi Biotec (8 mentions)
Cd3 percp, by Miltenyi Biotec (5 mentions)
Cd38 apc, by Miltenyi Biotec (5 mentions)
Macsquant analyzer flow cytometer, by Miltenyi Biotec (4 mentions)
Hla dr pe, by Miltenyi Biotec (4 mentions)
Macsquantify software 2, by Miltenyi Biotec (4 mentions)
Cd27 vioblue, by Miltenyi Biotec (3 mentions)
Cd45ro pe vio770, by Miltenyi Biotec (3 mentions)
Cd14 fitc, by Miltenyi Biotec (2 mentions)
Cd4 apc, by Miltenyi Biotec (2 mentions)
Cd3 pe vio770, by Miltenyi Biotec (2 mentions)
Cd3 vioblue, by Miltenyi Biotec (2 mentions)
Facscalibur, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Il 17 pe, by Miltenyi Biotec (1 mentions)
Cd4 pe, by Miltenyi Biotec (1 mentions)
Lsm710 confocal microscopy, by Zeiss (1 mentions)
Cd3 v500, by BD (1 mentions)
11 protocols using cd8 fitc
1
Immunophenotyping of Peripheral Blood
2
Intracellular Cytokine Production in CD4+ T Cells
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After separation with microbeads, CD4+ T cells were stained with mouse anti-human monoclonal antibodies: CD14-Fitc or CD8-Fitc, CD4-Pe, CD3-PercP and the isotype control (Miltenyi Biotec) and analyzed by flow cytometry to assess purity.
The analysis of intracellular cytokine production was done after 12 days of culture; T cells were stimulated for 6 hours with phorbol 12-myristate 13-acetate (PMA, 10 ng/mL), Ionomycin (500 ng/mL) and Brefeldin A (BFA 10 μg/mL) (Sigma Aldrich) and the intracellular staining was performed incubating T cells with mouse monoclonal antibody IL-17Pe (Miltenyi Biotec), T cells were acquired on a FACScalibur (BD Biosciences) and analyzed with Cell Quest software (BD Biosciences).
The analysis of intracellular cytokine production was done after 12 days of culture; T cells were stimulated for 6 hours with phorbol 12-myristate 13-acetate (PMA, 10 ng/mL), Ionomycin (500 ng/mL) and Brefeldin A (BFA 10 μg/mL) (Sigma Aldrich) and the intracellular staining was performed incubating T cells with mouse monoclonal antibody IL-17Pe (Miltenyi Biotec), T cells were acquired on a FACScalibur (BD Biosciences) and analyzed with Cell Quest software (BD Biosciences).
3
Internalization of MSC-Derived EVs by IECs
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To assess EV internalization by IECs, MSC membranes were stained with 2×10−6 M of PKH26 PKH26 Red Fluorescent dye (Sigma-Aldrich) according to manufacturer’s recommendations. Then, PKH26-labeled or -unlabeled MSCs were cultured in presence of IECs and EV uptake was assessed after 1, 2 or 4 days. At the end of co-culture, cells were detached by trypsin and stained with the following monoclonal antibodies: CD45-Vioblue (Miltenyi Biotec), CD3-V500 (BD Biosciences), CD4-APC-Vio770, CD8-FITC, CD14-FITC (Miltenyi Biotec), CD16-PercP-Cy5, CD19-PE-Cy7 (BD Biosciences) to identify the different IEC population, while TOPRO-3 was used to identify viable cells. The internalization of MSC-derived EVs by IECs was analyzed by FACS analysis.
To further confirm the transfer of EVs into IECs, cells were analyzed at the end of co-culture by confocal microscopy. Briefly, cells were detached by trypsin and stained with Viobright-FITC anti-human CD45 monoclonal antibody (Miltenyi Biotec). Then, cells were fixed using Cytofix/Cytoperm kit (BD Biosciences) and TOPRO-3 (Invitrogen Life Technologies) was used to reveal nuclei. Finally, cells were loaded into the CytoSpin centrifuge’s sample chamber and centrifuged 5 minutes at 400 rpm.
Images were obtained by LSM 710 confocal microscopy (Zeiss) at 63x magnification and elaborated with ZEN imaging software (Zeiss).
To further confirm the transfer of EVs into IECs, cells were analyzed at the end of co-culture by confocal microscopy. Briefly, cells were detached by trypsin and stained with Viobright-FITC anti-human CD45 monoclonal antibody (Miltenyi Biotec). Then, cells were fixed using Cytofix/Cytoperm kit (BD Biosciences) and TOPRO-3 (Invitrogen Life Technologies) was used to reveal nuclei. Finally, cells were loaded into the CytoSpin centrifuge’s sample chamber and centrifuged 5 minutes at 400 rpm.
Images were obtained by LSM 710 confocal microscopy (Zeiss) at 63x magnification and elaborated with ZEN imaging software (Zeiss).
4
Isolation of T Cell Subsets
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Following respirometry, cell suspensions were centrifuged and PBMC were resuspended in cell separation buffer (PBS, 0.5% bovine serum albumin, 2 mM EDTA; Miltenyi Biotec, Bergisch-Gladbach, Germany) for the separation of T cell subsets. Appropriate volumes of antibodies (CD4-APC [10 µl/107 cells], CD8-FITC [10 µl/107 cells], CD45RA-PE [5 µl/107 cells], CD3-PE-Vio770 [5 µl/107 cells], all purchased from Miltenyi Biotec) were added and cells were stained according to the manufacturer’s protocol. Naïve T helper cells (CD3+CD4+CD45RA+), memory T helper cells (CD3+CD4+CD45RA−), naïve cytotoxic T cells (CD3+CD8+CD45RA+), and memory cytotoxic T cells (CD3+CD8+CD45RA−) were then isolated by fluorescent-activated cell sorting on a BD FACSAria III cell sorter (BD Biosciences, Heidelberg, Germany). Propidium iodide staining was used to distinguish dead from living cells.
5
Flow Cytometry Analysis of PBMCs
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PBMC were collected and aliquoted in 1 × 106 cells/mL RPMI medium plus 10% Fetal Bovine Serum and then washed by centrifugation. The following anti-human monoclonal antibodies were added: CD3-PerCP, CD4+-APC-Vio770, CD8+-FITC, CD4+ 5RO-PEVio770, CD27-VioBlue, CD38-APC, (Miltenyi Biotec, Bergisch Gladbach, Germany). Samples were acquired by Miltenyi Biotec flow cytometer-MACSQuant Analyzer (8 fluorescence channels, 3 lasers). Gating and data analysis were performed using MACSQuantify software 2.5 (Miltenyi Biotec, Bergisch Gladbach, Germany).
6
Mouse T Cell Absolute Counts from Mandibular Vein
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T cell absolute count was performed on blood samples kept from the mandibular vein of the mouse.
For the phenotypic characterization of cell populations, the following antibodies were used: CD8-FITC (Miltenyi Biotec), CD25-APC (Pharmingen), CD3-PE-Vio770 (Miltenyi Biotec), CD4-APC-Vio770 (Milteny Biotec), CD45R (B220)-Violblu (Milteny Biotec), NK1.1-PE (Milteny Biotec).
At predetermined optimal concentrations, 100 μl of blood was stained by incubation with the antibodies. Fifty microliters of CountBright Absolute Counting Beads (Molecular Probes) was added, and, following lysis of red blood cells, cells were acquired on a CyAn Cytometer (Beckman Coulter). By comparing the ratio of bead events to cell events, absolute numbers of cells in the sample were calculated.
Some experiments were performed by acquiring the stained blood samples on the CytoFLEX cytometer (Coulter), equipped with a volumetric sample injection module, which enables volumetric sampling and provides absolute cell counts for all samples without the use of beads.
For the phenotypic characterization of cell populations, the following antibodies were used: CD8-FITC (Miltenyi Biotec), CD25-APC (Pharmingen), CD3-PE-Vio770 (Miltenyi Biotec), CD4-APC-Vio770 (Milteny Biotec), CD45R (B220)-Violblu (Milteny Biotec), NK1.1-PE (Milteny Biotec).
At predetermined optimal concentrations, 100 μl of blood was stained by incubation with the antibodies. Fifty microliters of CountBright Absolute Counting Beads (Molecular Probes) was added, and, following lysis of red blood cells, cells were acquired on a CyAn Cytometer (Beckman Coulter). By comparing the ratio of bead events to cell events, absolute numbers of cells in the sample were calculated.
Some experiments were performed by acquiring the stained blood samples on the CytoFLEX cytometer (Coulter), equipped with a volumetric sample injection module, which enables volumetric sampling and provides absolute cell counts for all samples without the use of beads.
7
Multiparameter Flow Cytometric Analysis
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Phenotypes and activation markers were evaluated by Miltenyi Biotec flow cytometer-MACSQuant Analyzer (8 fluorescence channels, 3 lasers) on freshly isolated PBMC. Immune activation was evaluated by multiparameter flow cytofluorimetric analysis by the following antihuman monoclonal antibodies: CD3-PerCP, CD4-APC-Vio770, CD8-FITC, CD45ROPE-Vio770, CD27-VioBlue, CD38-APC, and HLA-DR-PE (Miltenyi Biotec, Bergisch Gladbach, Germany).
8
Flow Cytometric Analysis of T-cell Subsets
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T-cells generated from different culture vessels were transferred into a 1.5 mL Eppendorf tube, centrifuged at 300×g for 10 min at room temperature. Cell pellet was resuspended in 100 µL of fluorophore solution and incubated in the dark for 20 min. The samples were washed repeatedly before resuspending in a final volume of 250 µL. MACSQuant Analyser 10 (Miltenyi Biotec) was used to acquire the data and MACSQuantify Software 2.11 (Miltenyi Biotec) was used for analyses. Antibodies used in analyses: Viobility 405/520 Fixable Dye (130-109-814), CD3-VioBlue (130-113-133), CD19-PE (130-113-646), CD56-PE-Vio770 (120-113-313), CD4-APC-Vio770 (130-113-251) and CD8-FITC (130-110-677) (Miltenyi Biotec).
9
Immunophenotypic analysis of T cells
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Peripheral blood samples were collected in tubes containing ethylenediaminetetraacetic acid and Lyse/Wash Procedure was applied; lymphocytes T cells enumeration and activation were evaluated by the following anti-human monoclonal antibodies: CD3 VioBlue, CD 4 APC-Vio770, CD8 FITC, CD38 APC and HLA-DRPE (MiltenyiBiotec). 5-color flow cytometric analysis was performed with the MiltenyiBiotec flow citometer-MACSQuantAnalyzer (8 fluoresces, 3 lasers). Gating analysis was developed with MACSQuantify software 2.5 (MiltenyiBiotec) with the same gating strategy applied to all samples. We tested specificity for the expression of CD38 and HLA-DR markers, by using Fluorescence Minus One method (FMO): each cell population was stained with all the fluorescence antibodies, except one, at a time, in order to verify whether in the absence of one antibody, the labelled cells were negative for the removed one; this method allows the subset to be gated and the background level for the subset to be determined.
10
Gut Lymphocyte Phenotyping and Cytokine Production
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Phenotypes and activation markers were evaluated by Miltenyi Biotec flow cytometer-MACSQuant Analyzer on isolated LPLs by the following antihuman monoclonal antibodies: CD3-PerCP, CD4-APC-Vio770, CD8-FITC, CD38-APC, and HLA-DR-PE (Miltenyi Biotec, Bergisch Gladbach, Germany). Gating analysis and data were analyzed using MACSQuantify software 2.5 (Miltenyi Biotec). To evaluate intracellular IL-17 and IFN-γ production, LPLs were cultured accordingly to our established protocol [27 (link)]. The following antihuman mAbs were used: CD3-PerCP, CD4-APC-Vio770, CD8-FITC, IL-17-PE, and IFN-γ-APC. The frequencies of gut CD4+ T LPLs (CD3+ CD4+) were calculated as ratio of the gut CD4+ T LPLs to total LPLs (CD4+ LPLs/total LPLs).
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