The induction of apoptosis in breast cancer cells via WA and SFN was quantitatively determined using flow cytometry and the Annexin V—conjugated Alexafluor 488 (Alexa 488) Apoptosis Vybrant Assay Kit (Life Technologies, Carsbald, CA, USA). After treatment, cells were harvested using the digestive enzyme trypsin. Upon detachment, cell pellets were collected via centrifugation. PBS wash buffer was used to wash pelleted cells twice, and after washing, cells were incubated with Alexa488 and propidium iodide (PI) for cellular staining in annexin binding buffer for 10 min in the dark at room temperature. The stained cells were analyzed by fluorescence-activated cell sorting (FACS) by using a FACS-Caliber instrument (BD Biosciences, San Jose, CA, USA) equipped with Cell Quest 3.3 software (BD Biosciences, San Jose, CA, USA).
Facs caliber instrument
Manufactured by BD
Sourced in United States
The FACS Caliber is a flow cytometry instrument manufactured by BD. It is designed to analyze and sort cells based on their physical and fluorescent characteristics. The core function of the FACS Caliber is to provide quantitative data on various cell populations within a sample.
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Lab products found in correlation
Cellquest software, by BD (3 mentions)
Test tube, by BD (2 mentions)
Monensin, by Merck Group (2 mentions)
Gallios instrument, by Beckman Coulter (2 mentions)
Phosflow lyse fix buffer, by BD (2 mentions)
Phosflow perm 3, by BD (2 mentions)
Cellquest 3, by BD (1 mentions)
Annexin 5 conjugated alexafluor 488 alexa 488 apoptosis vybrant assay kit, by Thermo Fisher Scientific (1 mentions)
Anti tcr β mab, by BioLegend (1 mentions)
Anti il 5 apc, by BioLegend (1 mentions)
Anti il 2 apc, by BioLegend (1 mentions)
Anti ifn γ fitc, by BD (1 mentions)
Foxp3 transcription factor staining buffer kit, by Cytek Biosciences (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Anti il4 pe, by BioLegend (1 mentions)
Annexin 5 apc solution, by Keygen Biotech (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Ab30667, by Abcam (1 mentions)
Ab12085, by Abcam (1 mentions)
Cd95 fas pe, by BD (1 mentions)
19 protocols using facs caliber instrument
1
Quantifying Apoptosis in Breast Cancer Cells
2
Intracellular Cytokine Staining of CD4+ T Cells
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The in vitro differentiated CD4+ T cells were stimulated using an immobilized anti-TCR-β mAb (3 µg/ml, H57–597, BioLegend) for 6 h in the presence of monensin (2 µM). The cells were fixed with 4% paraformaldehyde (Cat#163–20145, Wako) and permeabilized with permeabilization buffer (50 mM NaCl, 5 mM EDTA, 0.02% NaN3, and 0.5% Triton X-100). Then, the cells were stained using the following antibodies, anti-IL-4-PE (Cat#504103, BioLegend, 1:50), anti-IL-5-APC (Cat#504305, BioLegend, 1:50), anti-IL-13-PE (Cat#12–7133–41, eBiosience, 1:50), anti-IFN-γ-FITC (Cat#562019, BD Biosciences, 1:500), and anti-IL-2-APC (Cat#503809, BioLegend, 1:50). For the intracellular staining of Gata3, the Foxp3/Transcription Factor Staining Buffer Kit (cat#TNB-0607, TONBO) was used according to the manufacturer’s protocol. Flow cytometry was performed using a Gallios Flow Cytometer instrument (Beckman Coulter) and a FACS Caliber instrument (BD Biosciences) and the results were analysed using the FlowJo software program (Tree Star, Ashland, OR, USA).
3
Apoptosis Analysis of Huh-7 Cells
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After transfection with sh-Ctrl or sh-CDCA5 lentiviruses for 72 h, cells were stained with Annexin V-APC solution (KeyGEN, Jiangsu, China) according to the manufacturer’s instructions. Numbers of apoptotic Huh-7 cells were then determined using flow cytometry with a fluorescence-activated cell sorting (FACS) caliber instrument (BD). Percentages of apoptotic cells were calculated according to Annexin V staining.
4
Intracellular p-p38 MAPK Staining
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For intracellular p-p38 MAPK staining, 100 µl heparin-anticoagulated venous blood was stained with phycoerythrin-conjugated anti-CD14 monoclonal antibodies for 20 min at room temperature (1:10; cat. no 1543861; BD Biosciences). Cell fixation and permeabilization were performed using the BD cytofix/cytoperm kit according to the manufacturer's protocol (BD Biosciences). Subsequently, the cells were stained with FITC-conjugated anti-p-p38 MAPK monoclonal antibodies for 20 min at room temperature. (1:10; cat. no 127841; BD Biosciences). Then, the expression levels of p-p38 MAPK in CD14+ cells from patients were analyzed using a FACScaliber instrument (BD Biosciences).
5
Characterization of hTMSC Surface Antigens
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The specific surface antigens of hTMSCs were characterized by flow cytometry analysis. The cells were incubated into a test tube (BD, Franklin Lakes, NJ) at a density of 1×105 cells/ml, then washed three times with wash buffer (PBS and 3% FBS). The cells were incubated with primary antibody for 40 min with saturating concentrations of monoclonal antibodies CD14 (all anti-human CD from BD Biosciences, San Jose, CA), CD19, CD29, CD34, CD73, CD90, HLA-DR, TLR2 (all anti-human TLR from Abcam, Cambridge, ab9100), TLR3 (ab12085), TLR4 (ab30667), and TLR5 (ab13875). After the cells were washed three times in buffer and centrifuged at 1200 rpm for five minutes, they were resuspended in ice cold PBS and incubated with the secondary antibody for 30 min in the dark at 40°C. Cell fluorescence was evaluated by flow cytometry in a FACS Caliber instrument (BD) and the data were analyzed using Cell Quest software (BD).
6
Apoptosis Induction in Breast Cancer Cells
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Induction of apoptosis in human breast cancer cells caused by resveratrol and pterostilbene treatment alone or in combination was quantitatively determined by flow cytometry using Annexin V-conjugated Alexafluro 488 (Alexa488) Apoptosis Vybrant Assay Kit. Approximately 8 × 104 cells/2 ml were plated in 6-well plates. Medium containing freshly added resveratrol (15 μM), pterostilbene (5 μM) and combination of resveratrol + pterostilbene (15+5 μM, respectively) was added for 72 h. Following treatment, cells were collected from 6-well plates by trypsinization, washed with PBS, and incubated with Alexa488 and propidium iodide (PI) for cellular staining in Annexin-binding buffer at room temperature for 10 min in the dark. The stained cells were analyzed by fluorescence-activated cell sorting (FACS) by using a FACS-Caliber instrument (BD Biosciences) equipped with Cell Quest 3.3 software.
7
Cell Surface Antigen Expression Analysis
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Monoclonal antibodies specific for CD95 (Fas)-PE, CD54 (ICAM-1)-PE, CD1d-PE, and HLA-ABC-PE were obtained from BD: Becton, Dickinson and Company (San Jose, CA). The monoclonal antibody specific for CD273 (PL-L2)-PE was obtained from BioLegend (San Diego, CA). The cultured cells were stained for expression of cell-surface antigens and flow cytometry was performed using a FACSCaliber instrument (BD) and analyzed with CellQuest software (BD).
8
Cytokine Profiling of Transplant Immune Cells
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Cell cultures and the splenocytes and lung lymphocytes which were isolated from lung graft transplantation models were restimulated with PMA (0.25 mg/mL) and ionomycin (0.25 mg/mL) for 5 h and with brefeldin A (5 mg/mL) for 4 h, and then stained for surface CD4 and CD25. The cells were then fixed, permeabilized, and stained for IL-17A or FoxP3. Flow cytometry data were collected on a FACSCaliber instrument (BD Bioscience, San Jose, CA, USA) and analyzed with the FlowJo software (Tree Star Inc, San Carlos, CA, USA).
9
Intracellular Cytokine and Phospho-S6 Staining
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The cells were differentiated in vitro and stimulated with an immobilized anti-TCR-β mAb (3 μg/ml, H57-597; BioLegend) for 6 h with monensin (2 μM, cat#M5273; Sigma-Aldrich, St. Louis, MO, USA) for the intracellular staining of cytokines. Intracellular staining was then performed as described previously. For the intracellular staining of phosphorylated S6 ribosomal proteins, the CD8 T cells were fixed and permeabilized with BD Phosflow Lyse/Fix Buffer (cat#558049; BD Bioscience) and BD Phosflow Perm III (cat#558050; BD Bioscience) in accordance with the manufacturer’s instructions. Flow cytometry was performed using a FACS Caliber instrument (BD Biosciences) and Gallios instrument (Beckman Coulter, CA, USA), and the results were analyzed using the FlowJo software program (Tree Star, Ashland, OR, USA).
10
T Cell Cytokine and Phospho-S6 Analysis
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The cells were differentiated in vitro and stimulated with an immobilized anti-TCR-β mAb (3 μg/ml, H57-597; BioLegend) for 6 h with monensin (2 μM, cat# M5273; Sigma-Aldrich, St. Louis, MO, USA) for the intracellular staining of cytokines. Intracellular staining was then performed as described previously27 (link),52 (link). For the intracellular staining of phosphorylated S6 ribosomal proteins, the CD8 T cells were fixed and permeabilized with BD Phosflow Lyse/Fix Buffer (cat# 558049; BD Biosciences) and BD Phosflow Perm III (cat# 558050; BD Biosciences) in accordance with the manufacturer’s instructions. Flow cytometry (FACS) was performed using a FACS Caliber instrument (BD Biosciences) and Gallios instrument (Beckman Coulter, CA, USA), and the results were analyzed using the FlowJo software program (Tree Star, Ashland, OR, USA).
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