TM4 cell apoptosis after treatment with different MBPs was analysed using Annexin V-FITC and PI staining kits (Vazyme, Nanjing, China) according to the manufacturer’s requirements. Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences), and the data were analysed using Paint-A-Gate software (Becton-Dickson, San Jose, CA).
Paint a gate software
Manufactured by BD
Sourced in United States
Paint-A-Gate software is a digital tool designed for visualizing and configuring custom gate designs. It allows users to select from a variety of gate styles, materials, and colors to create a digital representation of their desired gate.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Pe conjugated anti cd45, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti cd34, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti cd31, by BioLegend (1 mentions)
Pe conjugated anti cd29, by BioLegend (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Cellquest software, by BD (1 mentions)
Anti human foxp3 apc, by Thermo Fisher Scientific (1 mentions)
Facscalibur cytometer, by BD (1 mentions)
Cd206 apc, by Thermo Fisher Scientific (1 mentions)
Facs aria 2 ow cytometer, by BD (1 mentions)
Facscalibur ow cytometer, by BD (1 mentions)
Cd11b fitc, by Thermo Fisher Scientific (1 mentions)
F4 80 pe, by Thermo Fisher Scientific (1 mentions)
Annexin 5 propidium iodide apoptosis kit, by BD (1 mentions)
Leucocount, by BD (1 mentions)
Truecount tubes, by BD (1 mentions)
12 protocols using paint a gate software
1
Quantifying TM4 Cell Apoptosis
2
Characterization of LR-MSC Surface Markers
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To determine the expression of various surface markers, LR-MSCs following the first passage were incubated with fluorescent antibodies at 37 °C for 40 min in the dark followed by two washes with PBS. Flow cytometry was performed on a FACS CaliburTM flow cytometer and the data were analyzed using the Paint-A-Gate software (Becton Dickinson). The antibodies used were: FITC-conjugated anti-Ly-6A/E (Sca-1), PE-conjugated anti-CD45 and PE-conjugated anti-CD34 (eBioscience, San Diego, CA); APC-conjugated anti-CD31 and PE-conjugated anti-CD29 (Bio-legend, San Diego, CA).
3
Apoptosis Analysis in Propofol-Treated Cells
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Cells after H/R and treatment with propofol (50 μM) or SP600125/Everolimus were harvested by brief trypsinization and centrifugation (at 170 ×g), washed in ice-cold PBS, and fixed in 70% ethanol for 2 hours at −20°C. Apoptosis was detected by double-staining with annexin V conjugated to fluorescein isothiocyanate and propidium iodide (Bender Medsystems). Ten thousand cells per sample were acquired in a FACScan flow cytometer and the proportions of labeled cells were analyzed using Paint-A-Gate software (Becton Dickinson).
4
Characterizing Macrophage Subtypes in Fibrotic Lungs
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To determine the macrophage subtypes in fibrotic lungs, lower right lungs were minced with fine scissors, and enzymatically digested with 0.1% type I collagenase (Sigma no. C0130) for 1 h at 37 °C with gentle agitation every 20 min. The single cell suspension was refiltered through a 40-μm nylon mesh after digestion to remove connective tissue, after which the cells were washed with D-Hanks and collected by centrifugation by 300×g for 5 min. Cells were incubated with PE-conjugated F4/80 antibodies or APC-conjugated CD206. Flow cytometry was performed on a FACS CaliburTM flow cytometer and the data were analyzed using Paint-A-Gate software (Becton Dickinson).
In addition, flow cytometric analyses were used to determine the macrophage polarization in vitro. RAW 264.7 cells after stimulation were incubated with fluorescent antibodies at 37 °C for 40 min in the dark followed by two washes with PBS. The antibodies used were: PE-conjugated anti-CD68, PE-conjugated anti-CCR7, and APC-conjugated anti-CD206.
In addition, flow cytometric analyses were used to determine the macrophage polarization in vitro. RAW 264.7 cells after stimulation were incubated with fluorescent antibodies at 37 °C for 40 min in the dark followed by two washes with PBS. The antibodies used were: PE-conjugated anti-CD68, PE-conjugated anti-CCR7, and APC-conjugated anti-CD206.
5
Flow Cytometric Immunophenotyping of BMSCs
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Passage 3 BMSCs were incubated with fluorescent-conjugated antibodies at 37°C for 1 h in the dark following two washes with phosphate-buffered saline (PBS, 1×). Cells were analyzed in a FACSCalibur flow cytometer using Paint-A-Gate software (Becton Dickinson, Franklin Lakes, NJ). The following antibodies were used: phycoerythrin (PE)-labeled rat anti-rat cluster of differentiation (CD) 29, CD31, CD34, CD44, CD45, and CD90 (Santa Cruz Biotechnology, Santa Cruz, CA).
6
Isolation and Characterization of Liver Cells
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Liver cells were isolated from liver fragments collected after animal sacrifice by using collagenase (collagenase-Hepatocyte-Qualified – Gibco/BRL) as described elsewhere48 (link). After liver cell isolation, viability and death, oxidative stress and mitochondrial membrane potential were evaluated by flow cytometry. Flow cytometry analysis was performed using a six-parameter, four-color FACS CaliburTM flow cytometer (Becton Dickinson) equipped with a 15 mW argon laser. For each assay 106 cells were used and at least 104 events were collected by acquisition using Cell Quest software (Becton Dickinson) and analysed using Paint-a-gate software (Becton Dickinson)13 (link)49 (link)50 (link)51 (link).
7
Comprehensive Immune Profiling of PBMC
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Blood samples, collected in pyrogen-free heparinized tubes (Biofreeze, Costar, EEUU), were taken at 8 am to minimize the influence of circadian rhythms. Peripheral blood mononuclear cells (PBMC) were obtained from heparinized venous blood by Ficoll-Hypaque (Lymphprep Nyegaard, Oslo, Norway) density gradient centrifugation. PBMC were incubated with combinations of fluorescein-CD127 (FITC), (Biolegend, San Diego CA, USA) phycoerythrin-CD25 (PE) (Becton-Dickinson, San Jose, CA, USA) and peridinin chlorophyll protein CD4 (PerCP) (Becton-Dickinson), fluorescein-CD45RA (FITC) (Becton-Dickinson), fluorescein-CD279 [–anti-PD1-] (FITC) (Becton-Dickinson), fluorescein-CD284 [–anti-TLR4-] (FITC) (Imgenex, San Diego CA, USA), and peridin chlorophyll protein CD14 (PerCP) (Becton-Dickinson) labelled monoclonal antibodies. The anti-human FOXP3 APC (eBioscience, San Diego CA, USA) was used for intracellular T cell immunophenotyping. Stained cells were washed, acquired, and analyzed by two-colour flow cytometry in a FACScalibur cytometer using Cell Quest and Paint-A-Gate software (Becton-Dickinson). FACS gating strategy and plots for all the cell subsets analysed are shown in Fig. 1 .
8
Phenotypic Analysis of Macrophages
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A phenotype analysis of the macrophages was performed with a BD FACS AriaII ow cytometer (BD, USA) as previously described [16] . Brie y, the cells were labeled with CD11b-FITC, F4/80-PE, and CD206-APC (eBioscience, San Diego, CA) following the manufacturer's protocol. To analyze the prevalence of M2 macrophages, the cells were incubated with PE-conjugated F4/80 antibodies or APC-conjugated CD206. Flow cytometry was performed using a FACS CaliburTM ow cytometer, and the data were analyzed using Paint-A-Gate software (Becton Dickinson).
9
Quantification of Cell Apoptosis
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To quantitate the amount of apoptosis, after harvesting cells which were incubated by various textures in complete medium, cells were washed twice with washing buffer (phosphate buffered saline 0.15 M, 0.5% bovine serum albumin, 0.1% NaN3). Fresh B-cells were resuspended in 100 µl washing buffer and stained with appropriate fluorochrome-conjugated mAbs and incubated for 45 min at 4°C in the dark atmosphere to evaluate the amount of apoptosis by using Annexin V/Propidium iodide apoptosis kit (BD Biosciences, San Jose, CA, USA). Then 1000 cells/sample was acquired in an FAC Scan flow cytometer (Partec, Nuremberg, Germany). Moreover, the proportions of labeled cells were analyzed using Paint-A-Gate software (Becton and Dickinson, Sunnyvale, USA). Data analysis was performed using the Flomax software (Partec, Nuremberg, Germany).
10
Flow Cytometric Analysis of Vimentin, CD44, and CD71
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The flow cytometry protocol used was similar to the immunofluorescence protocol described earlier, in terms of the cell preparation and treatment. However, the selected polyclonal antibodies used were as follows: Anti-vimentin, anti-CD44 and anti-CD71 antibodies (all rabbit anti-chicken antibodies; dilution, 1:100; cat. nos. bs-0756R, bs-2507R and bs-1782R, respectively; Beijing Biosynthesis Biotechnology Co. Ltd.). Flow cytometric analysis was performed on the mononuclear cell suspension, and 5×104 cells were incubated with 10 µl FITC-conjugated antibodies (18 (link)). Cells were acquired using a FACSCalibur flow cytometer and CytExpert Cell Quest software, and then analyzed with Paint-A-Gate software (BD Biosciences, San Jose, CA, USA).
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