The cell cycle kinetics of parental and transfected cells was determined using flow cytometry. For this, cells were harvested and washed with cold 1× PBS (Phosphate Buffered Saline) (HiMedia, Mumbai, MH India) buffer and fixed with 75% ethanol for 30 min on ice. Further, cells were washed 3 times with cold 1× PBS and incubated with 50 μg/mL of propidium iodide (PI) (Invitrogen) and 10 μg/mL of DNase-free RNase A in 500 μL of 1× PBS for 30 min at 37 °C. Later, cells were re-suspended in 500 μL of 1× PBS and acquired on FACSCalibur (BD Bioscience). Cells were acquired and DNA histograms were generated and analyzed by CellQuest™ Pro 5.2.1 software (BD Biosciences). For cell surface marker analyses for CD44 expression, cells were harvested from culture and washed in cold 1× PBS buffer and fixed with 75% ethanol for 30 min on ice. Further, cells were washed with cold 1× PBS and blocked with blocking buffer (5% BSA) for 30 min at 4 °C. Cells were incubated with primary antibody against CD44 (Chemicon International Billerica, MA, USA) for 1 h followed by incubation with secondary antibody Alexa Fluor 488 (Invitrogen). The cells were then washed with cold 1× PBS, re-suspended in 500 μL of cold 1× PBS and acquired on FACSCalibur (BD Bioscience). DNA histograms were generated and analyzed by CellQuest™ Pro 5.2.1 software (BD Bioscience).
Cellquest pro 5
Manufactured by BD
Sourced in United States
CellQuest Pro 5.1 is a software application designed for flow cytometry data analysis and management. It provides users with tools for acquiring, analyzing, and organizing flow cytometry data.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (13 mentions)
Facscalibur cytometer, by BD (8 mentions)
Facscalibur flow cytometer, by BD (6 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by HiMedia (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometry analysis, by BD (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Perm wash solution, by BD (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Pi for flow cytometry, by Thermo Fisher Scientific (1 mentions)
Vybrant apoptosis assay kit 4, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Monensin, by Merck Group (1 mentions)
Phorbol ester, by Merck Group (1 mentions)
Cytofix cytoperm solution kit, by BD (1 mentions)
36 protocols using cellquest pro 5
1
Cell Cycle Kinetics and CD44 Analysis
2
Cell Cycle Analysis by Flow Cytometry
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The exposure effect on the cell cycle was determined by flow cytometric analysis. At and 24 h after irradiation, adherent and suspended cells were harvested, centrifuged at 1500 rpm for 10 min, and washed twice with cold phosphate buffered saline (PBS). The assay was then performed, as previously described in Benvenuto et al. [92 (link)]. Cells were analyzed with flow cytometry using a FACS Calibur cytometer, running CellQuest Pro 5.2 software (BD Biosciences, San Jose, CA, USA).
3
Apoptosis Analysis of HK-2 Cells
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HK-2 cells of logarithmic growth phase were diluted in DMEM medium containing 10% fetal
bovine serum (FBS, Thermo Fisher Scientific, Inc., Waltham, MA, USA) to a final
concentration of 5×105 cells/mL. Cells were suspended in a concentration of
1×106 cells and incubated for 24 hours. Then, the cells were digested with
pancreatic enzyme (Beyotime Institute of Biotechnology, Shanghai, China). After 3 minutes,
the cells were harvested, washed with phosphate buffer saline (PBS, Sinopharm
Pharmaceutical Co. Ltd, Shanghai, China) and re-suspended in 1× kit binding buffer. Then,
the ratio of apoptotic cells was examined using an Annexin V-fluorescein isothiocyanate/PI
kit (BD PharMingen, San Diego, CA, USA). After, cells were incubated with Annexin V and PI
according to the manufacturer’s instructions for 15 minuts at 26˚C in the dark, subjected
to FACSCalibur flow cytometry analysis (BD Biosciences, San Jose, CA, USA), using Cell
Quest Pro 5.2 software (BD Biosciences).
bovine serum (FBS, Thermo Fisher Scientific, Inc., Waltham, MA, USA) to a final
concentration of 5×105 cells/mL. Cells were suspended in a concentration of
1×106 cells and incubated for 24 hours. Then, the cells were digested with
pancreatic enzyme (Beyotime Institute of Biotechnology, Shanghai, China). After 3 minutes,
the cells were harvested, washed with phosphate buffer saline (PBS, Sinopharm
Pharmaceutical Co. Ltd, Shanghai, China) and re-suspended in 1× kit binding buffer. Then,
the ratio of apoptotic cells was examined using an Annexin V-fluorescein isothiocyanate/PI
kit (BD PharMingen, San Diego, CA, USA). After, cells were incubated with Annexin V and PI
according to the manufacturer’s instructions for 15 minuts at 26˚C in the dark, subjected
to FACSCalibur flow cytometry analysis (BD Biosciences, San Jose, CA, USA), using Cell
Quest Pro 5.2 software (BD Biosciences).
4
Investigating Cell Cycle Effects of ERW
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Asynchronized, log-phase growing cells (60% confluent, approximately 2.5 × 105/well in 6-well plates) were treated with ERW, autoclaved ERW or control medium (CTR). Media were replaced every 24 hours. Fluorescence-activated cell sorting (FACS) analysis was performed as previously described using a FACSCalibur cytometer with CellQuest Pro 5.2 software (BD Biosciences, San Jose, CA) [26 (link), 31 (link)]. A minimum of 20000 events was collected. The experiments were repeated three times
5
Bortezomib-induced cell death assay
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Asynchronized log-phase growing cells (60% confluent, approximately 2.5 × 105 cells/well in 6-well plates) were treated with Bortezomib or with DMSO in a complete culture medium. Z-VAD-FMK was used at a final concentration of 40 µM for 2 h before addition of Bortezomib. After 48 h, adherent and suspended cells were harvested, centrifuged at 1500 rpm for 10 min and washed twice with cold phosphate buffered saline (PBS). The assay was then performed as previously described98 (link). Cells were analyzed by flow cytometry using a FACS-Calibur cytometer running CellQuest Pro 5.2 software (BD Biosciences, San Jose, CA, USA).
6
Cell Cycle Analysis of HDF Cells
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Log-phase growing HDF cells (60% confluent; 2.5×105/well in six-well plates) were treated with Galium or sodium chloride at the dilution of 1:2 in DMEM 0.2% BSA. Following a 48-h incubation, cells were harvested, centrifuged at 300 × g for 10 min at 4°C and washed twice with cold PBS. The cell pellets were re-suspended in 70% ethanol and incubated for 1 h at −20°C. The cells were then washed twice with cold PBS, centrifuged at 300 × g for 10 min at 4°C, incubated for 1 h in the dark with propidium iodide at a final concentration of 25 µg/ml in 0.1% citrate and 0.1% Triton X-100 (all Sigma-Aldrich; Merck KGaA). Samples were then analyzed by flow cytometry using a FACSCalibur™ cytometer with CellQuest Pro 5.2 software (both BD Biosciences) (21 (link)).
7
Flow Cytometry Analysis of Transduced Cells
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Forty-eight hours after retroviral transduction of genes in mouse primary T cells or Jurkat cell lines, the cells were washed in FACS buffer (1% BSA plus 0.1% sodium azide) and analyzed by flow cytometry (FACSCalibur using CellQuest Pro 5.2.1 software [BD Biosciences]). Data were analyzed with FlowJo 9.2 software (TreeStar).
8
Th1 and Th17 Cell Differentiation
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CD4+ T cells were stimulated with plate-bound anti-CD3 (2 µg/ml) and anti-CD28 (2 µg/ml) in Th0 (neutral), Th1 (10 ng/ml IL-12 and 10 µg/ml anti–IFN-γ), or Th17 (20 ng/ml IL-6, 5 ng/ml TGFβ, 10 µg/ml anti–IFN-γ, and 10 µg/ml anti–IL-4) conditions for 3 d. Skewed cells were placed in 96-well plates and restimulated with PMA (10 ng/ml) and ionomycin (1 µg/ml) in the presence of monensin (3 µM) for 4 h at 37°C. Cells were then washed in FACS buffer (1% BSA plus 0.1% sodium azide) and fixed and permeabilized with Cytofix/Cytoperm solution for 25 min on ice, followed by washing in Perm/Wash solution (BD). Cells were stained for 30 min at room temperature with mAbs against IL-17A and IFN-γ, washed two times in Perm/Wash solution, and once with FACS buffer, and analyzed by flow cytometry (FACSCalibur using CellQuest Pro 5.2.1 software [BD]). Data were analyzed with FlowJo 9.2 software (Tree Star).
9
Flow Cytometry Analysis of FRA1 Expression
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Cells were collected by trypsin and washed in ice-cold PBS. Cell number was adjusted to a concentration of 1 × 106 cells per sample. Additionally, after fixing for 15 min in 4% paraformaldehyde, cells were permeabilized in 0.3% Triton-X 100 (in PBS, pH 7.4) and blocked with 2% bovine serum albumin (BSA) in PBS. Cells were incubated with rabbit monoclonal Anti-FRA1 antibody conjugated with Alexa Fluor® 488 (dilution 1:500, clone EP4711, Abcam, Cambridge, MA, USA) for 1 h. Following the three cycles of washing with PBS containing 0.1 % BSA, cells were analyzed using BD FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). A total of 50,000 events were acquired per sample. Median fluorescence intensity was calculated with BD CellQuest Pro 5.1 software (Becton Dickinson, San Jose, CA, USA).
10
Quantify Early-Stage Apoptosis by Flow Cytometry
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To quantify the proportion of early-stage apoptotic cells the commercial kit “Vybrant Apoptosis Assay Kit #4” with YO-PRO-1 and PI for Flow Cytometry (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA; catalog number: V13243) was used. The cells were stained according to the supplemented manufacturer protocol. After 24 hours post-radiation, cells were collected and washed in cold phosphate-buffered saline (PBS). A total of 1 μL of YO-PRO-1 stock solution and 1 μL of PI stock solution were added to each 1 mL containing 1 × 106 cells. Cells were incubated on ice for 20–30 min. Cells were analyzed by flow cytometry (BD FACSCalibur, Becton Dickinson, San Jose, CA, USA) using 488 nm excitation with green fluorescence emission for YO-PRO-1 (i.e., 530/30 bandpass) and red fluorescence emission for PI (i.e., 610/20 bandpass). A total of 50,000 events were acquired for each sample and analyzed with BD CellQuest Pro 5.1 software (Becton Dickinson, San Jose, CA, USA).
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