Mice were fed with SCFAs for 30 days and were sacrificed by carbon dioxide asphyxiation, followed by extraction of spleen and colon tissues. Preparation of cell suspension by homogenization and adjust the cell concentration to 107/ml. Afterwards, cell suspension was incubated with 20 μl of PC5-CD3, FITC-CD4 and PE-CD8 monoclonal antibodies (BD, MA, USA) at room temperature in the dark for 15 min, washed with pre-cooled PBS and centrifuged at 1500rpm. After mixing with 0.5 ml of PBS, samples were subjected to EPICS XLII flow cytometer (Beckman Coulter, CA, USA), followed by calculation of CD3+CD4−CD8− cell ratio using CellQuest software.
Cd3 pc5
Manufactured by BD
Sourced in United States
The CD3-PC5 is a laboratory equipment product. It serves a core function as a measurement tool. No further details can be provided while maintaining an unbiased and factual approach.
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Lab products found in correlation
Cd4 fitc, by BD (2 mentions)
Epics xlii flow cytometer, by Beckman Coulter (2 mentions)
Cd4 pe, by BD (2 mentions)
Live dead near ir, by Thermo Fisher Scientific (1 mentions)
Cd11b pe, by BD (1 mentions)
Fortessa cytometer, by BD (1 mentions)
Cd19 pc5, by BD (1 mentions)
Cd10 pecf594, by BD (1 mentions)
Cd62l pe, by BD (1 mentions)
Cd15 fitc, by Thermo Fisher Scientific (1 mentions)
Live dead fixable aqua, by Thermo Fisher Scientific (1 mentions)
Cd16 af700, by BioLegend (1 mentions)
Lsrii sorp cytometer, by BD (1 mentions)
Lox 1 pe, by BioLegend (1 mentions)
Cd66b pe, by BD (1 mentions)
Pd l1 bv421, by BD (1 mentions)
Cxcr1 pe, by BD (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
Cd25 fitc, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
6 protocols using cd3 pc5
1
SCFA-Induced Immune Cell Profiling
2
Profiling Liver Fibrosis Immune Cells
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This study enrolled 25 liver fibrosis patients diagnosed at The Second Affiliated Hospital of Jiaxing University and 25 healthy subjects from the Physical Examination Center from January 2019 to January 2020. After informed consent was obtained from each patient, 8 ml of peripheral blood (elbow vein blood) was collected to extract peripheral blood mononuclear cells (PBMCs). Later, the samples were divided into two portions, one for the detection of DNT proportion and the other one for the detection of CD3+TNF-α+ proportion. Later, cells were incubated with monoclonal antibodies against PC5-CD3, FITC-CD4, and PE-CD8 (20 μl each; BD, Massachusetts, USA) at room temperature in dark for 15 min. After being washed with the pre-chilled phosphate-buffered saline (PBS), cells were centrifuged at 1,500 rpm, mixed with 0.5 ml of PBS, and detected using the EPICS XLII flow cytometer (Beckman-Coulter, California, USA), and the CD3+CD4−CD8− cell proportion was analyzed with CellQuest software. Moreover, when detecting DNTs with monoclonal antibodies against PC5-CD3, FITC-CD4, PE-CD8, and APC-TNF-α (20 μl each), the proportion of CD3+TNF-α+ in DNTs was analyzed by CellQuest software.
3
Multiparameter Flow Cytometry for Immune Cell Analysis
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Cell suspensions were stained with Near-IR LIVE/DEAD™ (Life Technologies), CD3-PC5 or CD19-PC5, and CD11b-PE when indicated (BD Biosciences). Samples were acquired on a FORTESSA cytometer (BD Biosciences). Data were exported and analyzed with FlowJo (RRID : SCR_008520; version 9-2, MacOS X). Counting beads and cells were gated on forward scatter-area/side-scatter area- (FSC-A/SSC-A) (shown in Figure 1C Figure S1A
4
Phenotypic Characterization of PMN-MDSCs
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Panel 1 included antibodies allowing the gating of PMN-MDSCs in the PBMCs and the analysis of expression of surface markers. It comprised of CD3-BV510, CD56-BV510, CD19-BV510, CD11b-APCH7, CD15-BV711, CD14-BV650, HLA-DR-BV605, CD10-PE-CF594 (Becton Dickinson), CD16-AF700 (BioLegend, Ozyme, Saint-Cyr-l’Ecole, France), CD33-PC7 (eBiosciences, Thermo Fisher), LIVE/DEAD fixable aqua (Life Technologies, Villebon-sur-Yvette, France). LOX-1-PE, CD11b-activated-PE (clone CBRM1/5) (Bio-Legend, Ozyme), CD62L-PE, CD66b-PE, PDL-1-BV421, CXCR1-PE, CD172ab-AF647 (BD Biosciences) were alternately included in Panel 1.
Panel 2 was used for functional analysis. It contained CD15-FITC (eBiosciences, Thermo Fisher), CD3-PC5 (BD Biosciences) and LIVE/DEAD near-IR (Live Technologies,). CD66b-PE, CD10-PE-CF594 and CD16-AF700 were occasionally added. PBMCs were stained 20 min at room temperature with fluorescent reagents pre-mixed in PBS, washed, then fixed with 4% paraformaldehyde and analyzed by FACS within 4 days on LSRII-SORP cytometer (BD Biosciences) equipped with four lasers (405 nm/100 mW, 488 nm/100 mW, 560 nm/50 mW and 630 nm/40 mW). PMT were set using unstained and fully stained samples. Compensations were performed with beads stained with corresponding reagents. Data were exported and analyzed with FlowJo (version 9-2, MacOS X).
Panel 2 was used for functional analysis. It contained CD15-FITC (eBiosciences, Thermo Fisher), CD3-PC5 (BD Biosciences) and LIVE/DEAD near-IR (Live Technologies,). CD66b-PE, CD10-PE-CF594 and CD16-AF700 were occasionally added. PBMCs were stained 20 min at room temperature with fluorescent reagents pre-mixed in PBS, washed, then fixed with 4% paraformaldehyde and analyzed by FACS within 4 days on LSRII-SORP cytometer (BD Biosciences) equipped with four lasers (405 nm/100 mW, 488 nm/100 mW, 560 nm/50 mW and 630 nm/40 mW). PMT were set using unstained and fully stained samples. Compensations were performed with beads stained with corresponding reagents. Data were exported and analyzed with FlowJo (version 9-2, MacOS X).
5
Multiparameter Flow Cytometry of Lymphocyte Subsets
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Peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll–Paque (Pharmacia) density-gradient centrifugation. After blocking with FcR, PBMCs were incubated with diluted antibodies for phenotyping and then analyzed using flow cytometry (FC-500, Beckman Coulter, FL, USA). Data analysis was conducted using FlowJo software (version 7.6.2, Tree Star, Ashland, OR, USA). Circulating T lymphocyte subgroup and NK cells were identified by multiparameter flow cytometry according to the previous description [18 (link), 19 (link)]. T lymphocyte subsets were identifying CD4-FITC/CD8-PE/CD3-PC5, and NK cell was identifying with CD3-FICT/CD(16+56)-PE (BD Biosciences, CA, USA). The phenotype of NK cells was CD3-CD16+CD56+. Four-color flow cytometry (FC-500, Beckman Coulter, FL, USA) was performed to determine the phenotypes of T regulatory cells (Tregs) using the CD3-PC5, CD4-PE, CD25-FITC (BD Biosciences, CA, USA), and CD127-PC7 antibodies (BioLegend, San Diego, CA, USA). Tregs were defined as CD3+CD4+CD25+CD127− cells.
6
Comprehensive Blood Profile Analysis
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4–5 mL of peripheral venous blood was collected immediately from all patients after admissionfor blood routine, C-reactive protein (CRP), liver function, myocardial enzyme spectrum, calcitonin, cytokines and other laboratory examinations. HP Sysmex xs-800i was used to test blood routine and CRP. Beckman AU5800 (Beckman Kurt LTD, USA) automatic biochemical analyzer was used to determine liver function and myocardial enzyme spectrum.
Serum IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ levels were determined using a CBA HumanTh1/Th2 Cytokine Kit II (BD Biosciences, San Diego, CA, USA) according to the manufacturer's specifications. Following the acquisition of sample data using a FACSCalibur flow cytometer (BD Biosciences), results were generated using BD CBA Software (BD Biosciences, San Jose, CA, USA). Then we established the standard curve for each reagent. 1.0 pg/mL was the lowest detection limit for these six cytokines, while 5000 pg/mL was the highest.
T-cell subsets were detected by multicolor flow cytometry (FACSCalibur, BD, USA) using blood samples containing heparin. Mouse anti-human CD45-FITC, CD3-PC5, CD4-PE, and CD8-ECD monoclonal antibodies, and other reagents were purchased from BD. Data was analyzed by the MultiTEST software.
Serum IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ levels were determined using a CBA HumanTh1/Th2 Cytokine Kit II (BD Biosciences, San Diego, CA, USA) according to the manufacturer's specifications. Following the acquisition of sample data using a FACSCalibur flow cytometer (BD Biosciences), results were generated using BD CBA Software (BD Biosciences, San Jose, CA, USA). Then we established the standard curve for each reagent. 1.0 pg/mL was the lowest detection limit for these six cytokines, while 5000 pg/mL was the highest.
T-cell subsets were detected by multicolor flow cytometry (FACSCalibur, BD, USA) using blood samples containing heparin. Mouse anti-human CD45-FITC, CD3-PC5, CD4-PE, and CD8-ECD monoclonal antibodies, and other reagents were purchased from BD. Data was analyzed by the MultiTEST software.
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