Anti-CD4 (GK1.5), anti-CD25 (PC-61), anti-Vα3.2 (RR3-16), anti-CD45.2 (104), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-ICOS (C398.4A), anti-PD-1 (29F.1A12) and anti-CD73 (TY/11.8) were from BioLegend. Anti-Foxp3 (FJK-16a) was from eBioscience. Cell sorting and analyses were performed on ARIA III, FACS CALIBUR, LSRII, and CANTO (BD). For detection of apoptosis, FITC Annexin V staining kit and Zombie Violet viability dye were used from BioLegend. For intracellular staining cells were fixed and permeabilized using Fixation/Permeabilization buffers from eBioscience and BD according to manufacturers’ manual.
Fitc annexin 5 staining kit
Manufactured by BioLegend
The FITC Annexin V staining kit is a laboratory reagent used to detect and quantify apoptosis, or programmed cell death, in a sample. Annexin V is a protein that binds to phosphatidylserine, a phospholipid that is exposed on the outer cell membrane during the early stages of apoptosis. The kit includes FITC-conjugated Annexin V, which allows the identification and enumeration of apoptotic cells using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Anti foxp3 fjk 16a, by Thermo Fisher Scientific (1 mentions)
Anti cd73 ty 11.8, by BioLegend (1 mentions)
Zombie violet viability dye, by BioLegend (1 mentions)
Fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Aria 3, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Ccl 81, by American Type Culture Collection (1 mentions)
Ccl 186, by American Type Culture Collection (1 mentions)
Cho k1, by American Type Culture Collection (1 mentions)
Ccl 61, by American Type Culture Collection (1 mentions)
Ht 29, by American Type Culture Collection (1 mentions)
Htb 38, by American Type Culture Collection (1 mentions)
Pyruvate kinase, by Merck Group (1 mentions)
3 protocols using fitc annexin 5 staining kit
1
Multiparameter Flow Cytometry Analysis
2
Annexin V-FITC Apoptosis Assay
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By passing 4 h from the irradiation, medium transfer was implemented and Annexin staining assay was conducted at the specific time of 24 h as an apoptosis induction appropriate interval [ 30 (link)
]. Annexin apoptotic assay was initiated by the use of FITC Annexin V Staining Kit (BioLegend), in accordance with the manufacturer’s instruction. Briefly, 5×105 cells were resuspended in 200 microliters of binding buffer (1×), and five microliters of Annexin V- FITC was added to each sample then incubated for 15 min at room temperature in the dark, followed by addition of 10 microliters of PI (20 μg/ml) as well. Samples were analyzed for the apoptotic and necrotic cells presence by the use of BD FACS Calibur flow cytometer (BD Bioscience). 10,000 calls per each sample were evaluated and the obtained data were analyzed using the BD Cell Quest Pro software.
]. Annexin apoptotic assay was initiated by the use of FITC Annexin V Staining Kit (BioLegend), in accordance with the manufacturer’s instruction. Briefly, 5×105 cells were resuspended in 200 microliters of binding buffer (1×), and five microliters of Annexin V- FITC was added to each sample then incubated for 15 min at room temperature in the dark, followed by addition of 10 microliters of PI (20 μg/ml) as well. Samples were analyzed for the apoptotic and necrotic cells presence by the use of BD FACS Calibur flow cytometer (BD Bioscience). 10,000 calls per each sample were evaluated and the obtained data were analyzed using the BD Cell Quest Pro software.
3
Isotopic Labeling of Glucose Metabolism
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Isotopically labeled glucoses (1-3H, 2-3H and 6-3H) were purchased from Perkin Elmer and 1-14C and 6-14C labeled glucose were purchased from American Radiolabeled Chemicals. H218O isotopically labeled water was purchased from Cambridge Isotope Laboratories. Hexokinase, pyruvate kinase, phosphoglucomutase, UDP-glucose pyrophosphorylase and inorganic pyrophosphatase were purchased from Millipore-Sigma. Isofagomine D-tartrate was purchased from Carbosynth. Deoxynojirimycin, castanopermine, swainsonine and australine hydrochloride were purchased from GlycoFine chemicals. D-gluconon-1,5- lactone was purchased from Frontier Scientific. Uridine disphsophoglucose disodium salt (UDP-glucose) was purchased from Abcam. Uridine 5’diphosphate disodium salt hydrate (UDP) was purchased from Millipore-Sigma. Vero, IMR90, CHO-K1 and HT-29 cells were purchased from ATCC (CCL-81, CCL-186, CCL61 and HTB-38). Full-length TcdA and TcdB toxins were purchased from List Biological Laboratories Inc. FITC-Annexin V staining kit was purchased from Biolegend. All other reagents were of analytical grade and purchased from commercial sources.
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