After stimulation, PBMCs and blood-derived monocytes were cultured in RPMI-1640 medium with 10% FBS and resuspended at a density of 0.5×105 cells per ml. The cells were then stained with directly conjugated antibodies for 30 min at 4°C in flow tubes, and then fixed with PBS containing 4% paraformaldehyde. The antibodies used for flow cytometry included anti-human CD16-APC (cat. no. 561304), anti-human CD14-PeCy7 (cat. no. 557154), anti-human CD206-APC-Cy7 (cat. no. 551136) and anti-human 163-Alexa Fluor 488 (cat. no. 560459) (all BD Biosciences). For the flow cytometry analysis, the cells were collected according to the multi-color flow cytometer (BD Biosciences), and the FACS data were analyzed using FlowJo 10.0 software (BD Biosciences). For monocyte sorting, CD14high or CD16high monocytes were isolated from PBMCs according to standard protocols.
Multicolor flow cytometer
Manufactured by BD
Sourced in United States
The Multicolor flow cytometer is a laboratory instrument designed to analyze and sort cells or other particles in a fluid sample. It can simultaneously detect and measure multiple fluorescent markers or labels on individual cells, enabling the identification and characterization of different cell populations within a complex sample.
Automatically generated - may contain errors
Lab products found in correlation
Fixation and permeabilization buffer kit, by BD (1 mentions)
Foxp3 alexafluor488, by BD (1 mentions)
Anti human ifn γ pe cy7, by BD (1 mentions)
Cell stimulation cocktail, by BD (1 mentions)
Fitc anti mouse cd4 antibody, by BioLegend (1 mentions)
Pe anti mouse ifn γ antibody, by BioLegend (1 mentions)
Fixation permeabilization solution kit, by BD (1 mentions)
Pe anti mouse il 4 antibody, by BioLegend (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Annexin 5 pi detection kit, by BD (1 mentions)
Fc500 flow cytometer, by Tree Star (1 mentions)
100 μm filter, by BD (1 mentions)
Stain buffer, by BD (1 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (1 mentions)
Fixation permeabilization solution, by BD (1 mentions)
6 protocols using multicolor flow cytometer
1
Multiparametric flow cytometry of human monocytes
2
Characterization of Immune Cell Subsets
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Cells from BALF and MH-S cells were examined by flow cytometry. The amount of M1 macrophages (CD80+), M2 macrophages (CD206+) and eosinophils (CCR3+) were identified with specific antibodies and analyzed by multicolor flow cytometer (BD Celesta, New-Jersey, United States). The mean fluorescence intensity (MFI) was calculated by FlowJo software version 10 (Stanford, California, United States).
3
Flow Cytometry Analysis of PBMC Cytokines
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We obtained PBMCs from patients with high or low levels of TSLP by density centrifugation using Ficoll‐Paque and cultured these cells in RPMI 1640 supplemented with 10% FBS. Cells were resuspended at a density of 0.5 × 105 cells·mL−1, and 100–200 μL of cell suspension was stimulated with cell stimulation cocktail (plus protein transport inhibitors; 1.5 μL·mL−1 of cell suspension; BD Biosciences). After stimulation in vitro, these cells were permeabilized with fixation and permeabilization buffer kit (BD Biosciences) and stained for 30 min at 4 °C in flow tubes with directly conjugated antibodies. The cells were fixed with 4% paraformaldehyde. The antibodies used for the flow cytometry analysis included anti‐human CD4‐allophycocyanin (560158; BD Biosciences), anti‐human IFN‐γ‐PeCy7 (557844; BD Biosciences), anti‐human IL‐4 (554516; BD Biosciences), anti‐human forkhead box P3 (Foxp3)‐Alexa Fluor 488 (560459; BD Biosciences) and anti‐human IL‐10‐phycoerythrin (554498; BD Biosciences). The cells were separated with a multicolor flow cytometer (BD Company, Franklin lakes, NJ, USA), and the fluorescence‐activated cell signaling data were analyzed by flowjo 10.0 software (BD Bioscience, San Jose, CA, USA).
4
Evaluating SARS-CoV-2 T Cell Responses
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Antigen-specific T cell immune responses were assayed on a multicolor flow cytometer (BD Biosciences). After collecting the splenocytes from immunized or unimmunized mice, 2 × 106 cells (100 μL) per sample were stimulated with the SARS-CoV-2 S-protein peptides for 4 h at 37 °C. Brefeldin A (Thermo Scientific) was then added into splenocytes and incubated for 6 h. Stimulated cells were washed in PBS/0.5% BSA and stained with APC/Fire 750 anti-mouse CD3 antibody (BioLegend, San Diego, CA, USA), FITC anti-mouse CD4 antibody (BioLegend), and Brilliant Violet 510 anti-mouse CD8a antibody (BioLegend) surface markers. The cells were then fixed using a Fixation/Permeabilization Solution Kit (BD Biosciences) and stained with PE anti-mouse IFN-γ antibody (BioLegend), PE anti-mouse IL-2 antibody (BioLegend), and PE anti-mouse IL-4 antibody (BioLegend). Data were analyzed with FlowJo software.
5
Apoptosis and Cell Cycle Analysis of Lung Cells
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For apoptosis and cell cycle analysis, 2 × 105 MLE-12 cells and MenSCs were seeded into the lower and upper transwell chambers of 6-well plates, respectively. An Annexin V/PI detection kit (BD Biosciences, CA, USA) was used to detect MLE-12 apoptosis, and a Cell Cycle and Apoptosis analysis kit (Beyotime Biotechnology, Haimen, China) was used to detect the MLE-12 cell cycle. Both experiments were performed according to the manufacturer’s instructions using a FC500 flow cytometer, and the data were analyzed using FlowJo software (Tree Star, OR, USA).
To determine the number of lung epithelial cells in lung tissues, flow cytometry was performed. The lung tissues were cut into small pieces and digested with type 1 collagenase for 1 h at 37 °C. Then a single-cell suspension was obtained by grinding the digested tissue through a 100-μm filter (BD Biosciences, CA, USA) before being centrifuged at 300×g for 10 min at 4 °C, and red blood cells were lysed twice. Subsequently, the cells were resuspended with stain buffer (BD Biosciences) and incubated with antibodies listed in Additional file5 , Table S1 for 30 min at 4 °C. Subsequently, the cells were washed twice with stain buffer before being analyzed with a multicolor flow cytometer (BD Biosciences). The data were analyzed using FlowJo software.
To determine the number of lung epithelial cells in lung tissues, flow cytometry was performed. The lung tissues were cut into small pieces and digested with type 1 collagenase for 1 h at 37 °C. Then a single-cell suspension was obtained by grinding the digested tissue through a 100-μm filter (BD Biosciences, CA, USA) before being centrifuged at 300×g for 10 min at 4 °C, and red blood cells were lysed twice. Subsequently, the cells were resuspended with stain buffer (BD Biosciences) and incubated with antibodies listed in Additional file
6
Investigating MSC Phenotypes via YAP and OPN
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PDLF and
BMMSC at the third passage were incubated on scaffolds or TCP in growth
medium. At 5 days post incubation, cells were processed for fixation
and permeabilization using a fixation/permeabilization solution and
perm/wash buffer (BD Biosciences). After washing, cells were stained
with an MSC-specific surface marker, CD105, in combination with and
without anti-YAP and anti-OPN antibodies. Cell populations positively
expressing YAP, OPN, CD105, or all of them were analyzed using a multicolor
flow cytometer (BD Biosciences) and FlowJo software.
BMMSC at the third passage were incubated on scaffolds or TCP in growth
medium. At 5 days post incubation, cells were processed for fixation
and permeabilization using a fixation/permeabilization solution and
perm/wash buffer (BD Biosciences). After washing, cells were stained
with an MSC-specific surface marker, CD105, in combination with and
without anti-YAP and anti-OPN antibodies. Cell populations positively
expressing YAP, OPN, CD105, or all of them were analyzed using a multicolor
flow cytometer (BD Biosciences) and FlowJo software.
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