Apoptosis was measured using Annexin V/PI co-staining. HCT116wt cells (1.5 × 105/ml) were pretreated with IPA-3 (10 μM) for 1 hour then treated with TQ (40 μM) for 24 hours. After collection, cells were centrifuged at 200 g for 5 min, 4°C and washed with 1X PBS. The pellet was resuspended in 100 μl Annexin-V-Fluos labeling solution (10 μl annexin reagent and 10 μl PI solution in 150 μl incubation buffer (according to manufacturer, Roche). The samples were incubated for 7 min in the dark, at room temperature then 100 μl incubation buffer was added. The cellular fluorescence was then measured using a Fluorescence Activated Cell Sorter (FACS) flow cytometer (BDFACS CantoTM II). Each sample was collected as 20,000 ungated events and the different cell populations were determined using FlowJo software (FlowJo7.6.5).
Incubation buffer
Manufactured by Roche
Sourced in Germany, United States
Incubation buffer is a laboratory reagent used to maintain the optimal pH and ionic environment for various biological assays and cell culture applications. It provides a stable and controlled environment to facilitate the incubation and growth of cells or the performance of enzymatic or biochemical reactions.
Automatically generated - may contain errors
Lab products found in correlation
Facscantotm 2, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Brdu incorporation assay kits, by Merck Group (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Fluorophore conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bromodeoxyuridine (brdu), by Nikon (1 mentions)
5 bromo 2 deoxy uridine labeling and detection kit 1, by Roche (1 mentions)
Fluoromount g plus dapi, by Southern Biotech (1 mentions)
Eclipse te2000 u inverted fluorescent microscope, by Nikon (1 mentions)
Bromodeoxyuridine (brdu), by Roche (1 mentions)
5 protocols using incubation buffer
1
Apoptosis Induction Assay using Annexin V/PI
2
Apoptosis Induction Assay for Prostate and Glioblastoma
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Capability of the selected scFv in inducing apoptosis in the prostate and glioblastoma cells were investigated by Annexin-V/propidium iodide (PI) assay. In total, 8×10
5cells were seeded per culture plate and incubated overnight at 37°C. The cells were treated with anti-RTF scFv antibody (1000 scFv/cell) for 24 h. Untreated cells were considered as negative control. The cells were harvested using 0.25% trypsin/EDTA, washed with cold PBS and transferred into flow cytometry tubes followed by adding Annexin V-FITC and PI to the both treated and untreated cells. Preparation was completed by adding incubation buffer (Roche Applied Science, Germany) to each tube. The tubes belonged to the 5 cell lines were read with BD FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed by WinMDI 2.5 software.
5cells were seeded per culture plate and incubated overnight at 37°C. The cells were treated with anti-RTF scFv antibody (1000 scFv/cell) for 24 h. Untreated cells were considered as negative control. The cells were harvested using 0.25% trypsin/EDTA, washed with cold PBS and transferred into flow cytometry tubes followed by adding Annexin V-FITC and PI to the both treated and untreated cells. Preparation was completed by adding incubation buffer (Roche Applied Science, Germany) to each tube. The tubes belonged to the 5 cell lines were read with BD FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed by WinMDI 2.5 software.
3
Cell Proliferation Assay with Brdu
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The proliferative capacity of the transfected cell was assessed using the bromodeoxyuridine (Brdu) incorporation assay kits (Millipore, Billerica, MA, USA). Briefly, cells (1×104 (link)) were seeded in each well of a 96-well plate after 48 hours of transfection. 10 µL of Brdu was subsequently added to each well. After 5 hours incubation, cells were fixed by FixDenat for 30 minutes. Cells were then incubated with anti-Brdu antibody in incubation buffer from Roche for 1 hour at 37°C. Cell nuclei were stained with 1 µM SYTOX Green (S7020, Life Technologies) at room temperature for 15 minutes. In each treatment, cells were washed with PBS at least three times.
4
Cell Cycle Analysis via BrdU and Cyclin D1
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Cells were synchronized as described above. After mitotic detachment, floating cells were seeded on poly‐l ‐lysine‐coated coverslips and grown in nocodazole‐free medium. Cells were cultured in 50 μm BrdU‐supplemented media for 45 min prior to fixation in cold‐fixing solution (70% ethanol, 15 mm glycine; pH 2.0). Cells were rinsed twice with PBS 1X and blocked in 3% BSA‐PBS 1X solution. Coverslips were incubated with 1:20 anti‐BrdU antibody or 1 : 100 anti‐cyclin D1 (M20, #SC‐718) in incubation buffer (Roche Applied Science, Indianapolis, IN, USA) for 40 min at 37 °C. Next, cells were incubated with 1 : 500 fluorophore‐conjugated secondary antibody (Molecular Probes, Invitrogen) in 1% BSA‐PBS 1X solution. Cells were PBS‐rinsed again, and nuclei were stained with Hoechst (Bio‐Rad).
5
Evaluating Muscle Stem Cell Proliferation
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To assess the proliferation of MuSCs and FAPs, 2.5 mg BrdU (Roche, Basel, Switzerland) was injected intraperitoneally (100 mg/kg) in PBS with a 29-gauge insulin syringe 24 h before cell isolation. For detection of BrdU, isolated cells were plated on collagen-coated six-well chamber slides at 37 °C overnight, following FACS isolation. Cells were briefly washed with PBS and processed with the 5-Bromo-2’-deoxy-uridine Labeling and Detection Kit I (Roche), according to the manufacturer’s instructions. Briefly, cells were fixed [70% ethanol, 30% 50 mM glycine (pH 2, Santa Cruz)] for 20 min at room temperature, washed for 10 min with PBS, and blocked with blocking buffer (20% goat serum/0.3% Triton-X in PBS) for 1 h at room temperature. Cells were stained with anti-BrdU antibody provided with the kit in Incubation buffer (1:10; Roche) for 30 min at 37 °C. After washing with PBS, Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (1:1000 in blocking buffer) was added and incubated in the dark for 1 h at room temperature. Cells were washed, chambers removed, and coverslips were mounted with fluoromount G plus DAPI (SouthernBiotech, Birmingham, AL). Cells stained for BrdU were imaged using an Eclipse TE2000-U inverted fluorescent microscope (Nikon, Minato City, Tokyo, Japan) and BrdU-positive cells were quantified in Image J.
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