Cell surface marker analysis was performed by flow cytometry (Miltenyi Biotec) to confirm the stem cell characteristics of ADSCs. The cell surface markers used were CD73 allophycocyanin (APC), CD90 fluorescein isothiocyanate (FITC), and CD105 peridinin-chlorophyll-protein (PerCP) Cy5.5 as positive MSCs markers, and lineage negative marker-PE including CD34, CD45, CD11b, CD19, and human leukocyte antigen (HLA)-DR (Becton Dickinson) as positive haematopoietic cells markers. The cells (1 × 105, passage 3) were stained with fluorescence-labelled probes specific to cell surface molecule. Data were obtained from 10,000 events per analysis.
Flow cytometry
Manufactured by Miltenyi Biotec
Sourced in Germany
Flow cytometry is a laser-based technology used to analyze the physical and chemical characteristics of particles, typically cells, within a fluid sample. It measures multiple parameters of a single cell as it passes through a laser beam, providing detailed information about the cell's size, granularity, and fluorescence properties.
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Lab products found in correlation
Macsquantify software, by Miltenyi Biotec (3 mentions)
Hla dr, by BD (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Streptavidin apc, by Southern Biotech (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Dmem medium, by GE Healthcare (1 mentions)
Fetal calf serum (fcs), by Lonza (1 mentions)
Zeocin, by InvivoGen (1 mentions)
Igg1 pe, by BD (1 mentions)
Igg2a fitc, by BD (1 mentions)
Anti cd16 32, by BD (1 mentions)
Apc conjugated anti mouse cd45, by BD (1 mentions)
Anti cd4 apc, by Thermo Fisher Scientific (1 mentions)
Anti cd8 pe, by Thermo Fisher Scientific (1 mentions)
Stemflow hmsc analysis kit, by BD (1 mentions)
Z vad fmk, by Merck Group (1 mentions)
Annexin 5, by BD (1 mentions)
Fitc labeled annexin 5, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
27 protocols using flow cytometry
1
Flow Cytometry Analysis of ADSCs
2
Isolation and Characterization of Splenic Immune Cells
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Spleens from pregnant mice at E14.5 were enzymatically digested and passed through a nylon tissue strainer to obtain a single-cell suspension. Red blood cells were lysed by 3.5% NaCl. Spleen leucocytes were obtained by density gradient centrifugation (Lympholyte®-M; 1400 × g; 30 min). Splenocytes or uNK cells were incubated for 30 min at 22 °C with the following antibodies: anti-CD4-APC, anti-CD3e-APC, and anti-IL17-PE (splenocytes) or DBA-Alexa-488, NKp46-Vioblue and CD107-APC (uNK). Cells were analyzed by flow cytometry (Miltenyi), at least 20,000 events were scored and analyzed with Flow Jo software. Single cells were gated for CD4 (Vioblue) and CD3e (APC) to select double-positive cells. Then, these double-positive cells were gated on PE for IL17. The percentage of CD4+CD3e+ IL17+ cells corresponds to triple-positive cells from the total splenocyte preparations (Supplementary Fig. 4 ) . Single cells were gated on Alexa-488 to select DBA+ cells, and then these cells were gated on Vioblue and APC to select NKp46 and CD107 double-positive cells CD107.
3
Allicin Induces Apoptosis in SKOV3 Ovarian Cancer Cells
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SKOV3 cells (2 × 104) were seeded in each well of 96-well plates and incubated at various concentrations of allicin for different periods. After treatment, the proliferative potential of the cells was analyzed using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer's protocol. For apoptosis assay, the SKOV3 cells were grown to approximately 75% confluence in 6-well plates and then treated with or without allicin (25 μg/mL, 48 h) and/or JNK inhibitors (20 μM, 30 min). After treatment, the cells were collected, washed, and resuspended in 200 μL of binding buffer at 2 × 105 cells/mL. The samples were subsequently incubated with 2.5 μL of Annexin V-FITC and 5 μL of propidium iodide in the dark for 15 min at room temperature and then analyzed by flow cytometry (Miltenyi, Germany).
4
Analysis of CD4 T Cell Population
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For analysis of CD4 T cell population after challenge studies, PBMCs were prepared from the whole blood of immunized and control chickens on day 5 post-challenge, and incubated on ice with biotinylated mouse Anti-chicken CD4 (CELL LAB, USA) followed by streptavidin-APC (Southern Biotech, USA) as per the manufacturer's instructions. A Miltenyi Biotec flow cytometry (Miltenyi Biotec, Germany) was used to collect 25,000 events after creating an appropriate live gate. FACS data were analyzed by MACSQuantify software (Miltenyi Biotec, Germany).
5
Isolation of CD271+ Osteosarcoma Cells
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CD271+ and CD271- OS cells were sorted using CD271 MicoBead Kit (Order no. 130-092-819, Miltenyi Biotec. Auburn, CA) depending on its manual. Briefly, OS cells were collected and dissociated into single-cell suspension with MACS separation Buffer. OS cells were incubated with FcR Blocking Reagent and CD271-PE antibody in 4 °C for 10 min. Then, FcR Blocking Reagent and Anti-PE MicroBeads were added and mixed at 4 °C for 15 min. After washed with PBS, CD271+ OS cells were sorted with magnetic separation columns and MACS separator. The quality of MACS was checked with flow cytometry (Order no.130-110-115, Miltenyi Biotec.) on a BD Fluorescence Activated Cell Sorting (FACS) Calibur machine (BD Biosciences, San Jose, CA).
6
Quantifying Intracellular ROS Levels
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The fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA, Sigma) was applied to evaluate the intracellular ROS levels. Cells were grown in six-well plates, and after being treated with different concentrations of AB38b or Res, the cells were incubated with 10 μM DCFH-DA at 37 °C for 25 min. Then, the distribution of DCF fluorescence in 10 000 cells was measured by flow cytometry (Miltenyi Biotec, Bergisch Gladbach, Nordrhein-Westfalen, Germany) at an excitation wavelength of 488 and an emission wavelength of 525 nm.
7
Cell Cycle Analysis by Flow Cytometry
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ImGIST and PriGIST cells were washed with pre-cooled PBS and fixed overnight in 70% ethanol at 4 °C. The next day, cells were resuspended in 500 μL propidium iodide/RNase A staining solution (Beyotime) and then warmed to 37 °C for 30 min in the dark. The cell cycle was detected using flow cytometry (Miltenyi Biotec, Bergisch Gladbach, Germany). The proliferation index (PI) was calculated as follows: PI = (S + G2/M)/(G0/G1 + S + G2/M).
8
Establishment of Stable HEK293 Cell Lines
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HEK293 cells (LGC Standards (ATCC), Wesel, Germany) were maintained in DMEM medium (PAA Laboratories, Pasching, Austria) supplemented with 10% [v/v] FCS (Lonza, Basel, Switzerland), penicillin (100 units/ml)/streptomycin (100 µg/ml) and 2 mM L-glutamine at 37°C in a 7% [v/v] CO2 environment. Confluent cell layers were split twice a week.
Cells at 60–80% confluency were transfected with pcDNA3 plasmids containing the coding sequences for either myc-tagged mH1R or Flag-tagged mH4R, or the empty vectors using Fugene HD (Roche, Mannheim, Germany) according to the manufacturer's protocol. Cells with stable integration of vectors or plasmids were selected with G418 (empty vector, mH1R) or Zeocin (empty vector, mH4R) (both Invivogen, San Diego, CA, USA). Clones were generated by limiting dilution technique and receptor expression was checked by flow cytometry (Miltenyi Biotech, Bergisch Gladbach, Germany) and Western blot using anti-Flag and anti-myc (Roche,Penzberg, Germany) reagents.
Cells at 60–80% confluency were transfected with pcDNA3 plasmids containing the coding sequences for either myc-tagged mH1R or Flag-tagged mH4R, or the empty vectors using Fugene HD (Roche, Mannheim, Germany) according to the manufacturer's protocol. Cells with stable integration of vectors or plasmids were selected with G418 (empty vector, mH1R) or Zeocin (empty vector, mH4R) (both Invivogen, San Diego, CA, USA). Clones were generated by limiting dilution technique and receptor expression was checked by flow cytometry (Miltenyi Biotech, Bergisch Gladbach, Germany) and Western blot using anti-Flag and anti-myc (Roche,Penzberg, Germany) reagents.
9
Quantification of Endothelial Progenitor Cells
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To quantify EPCs number, cells from peripheral blood, BM homogenates and splenic tissue homogenates were lysed and used for flow cytometric analysis. All procedures were performed according to the manufacturer's instructions. After 30 min incubation with FITC-conjugated anti-mouse CD34 (BD Biosciences), PE-conjugated anti-mouse Flk-1 (BD Biosciences) and APC-conjugated anti-mouse CD45 (BD Biosciences), cells were washed with PBS and fixed in 4% paraformaldehyde and analyzed by Flow Cytometry (Miltenyi Biotec, Bergisch Gladbach, Germany). Staining was performed in the presence of saturating concentrations of rat monoclonal unconjugated antibodies against Fc receptors (anti-CD16/32, BD Bioscience) to reduce nonspecific binding. Isotype-identical antibodies served as controls (IgG1-PE and IgG2a-FITC, BD Bioscience). Each analysis included 100,000 events. Data were analyzed using MACSQuantify Software (Miltenyi Biotec). The number of CD34/Flk-1 double positive and CD45 negative cells was counted as EPCs.
10
Quantifying T-cell Infiltration in Blood, Liver, and Spleen
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Single-cell suspensions of blood, liver, and spleen were obtained 12 h after Con A administration. Cells were then stained with fluorescence-labeled antibody (anti-CD4 APC, code: 17-0041-82; clone: GK1.5, eBioscience USA, anti-CD8 PE, code: 11-0081-85; clone: 53-6.7, eBioscience USA). The counts of CD4+ and CD8+ T lymphocytes infiltrating in the blood, liver, and spleen were analyzed by flow cytometry (Miltenyi, Germany).
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