Transfected cells were collected 48 h after transfection, and then the cells were fixed with pre-cooled 70% ethanol, and incubated overnight at 4°C. Subsequently, the cells were washed once with 1 ml PBS, followed by incubation in the dark with 500 µl PBS containing 50 µg/ml propidium iodide, 0.2% Triton X-100 and 100 µg/ml RNase A for 30 min at 4°C. The cell cycle was analyzed using a flow cytometer and the Modfit LT version 4.1 software (Verity Software House, Inc.).
Modfit lt version 4
Manufactured by Verity Software House
Sourced in United States
ModFit LT version 4 is a software tool for cell cycle and DNA content analysis. It provides tools for DNA histogram analysis, cell cycle modeling, and data reporting.
Automatically generated - may contain errors
Lab products found in correlation
Accuri c6, by BD (1 mentions)
Pi rnase staining buffer, by BD (1 mentions)
Fortessa, by BD (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Nanog, by Cell Signaling Technology (1 mentions)
Canto flow cytometer, by BD (1 mentions)
Annexin 5, by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Mouse cell depletion kit, by Miltenyi Biotec (1 mentions)
Facsverse, by BD (1 mentions)
Cycletest plus dna reagent kit, by BD (1 mentions)
7 protocols using modfit lt version 4
1
Cell Cycle Analysis by Flow Cytometry
2
Cell Cycle Analysis by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells (1 × 106) were harvested at 48 hrs after transfection, washed once with PBS and fixed in 70% ethanol (diluted with PBS) at 4°C overnight. Then the cells were harvested, washed once with PBS again and incubated with 0.5 ml staining fluid containing 50 μg/ml propidium iodide, 0.1 mg/ml ribonuclease A and 0.2% Triton X‐100 for 30 min. at 4°C. Cell‐cycle distribution was measured by BD Accuri C6 flow cytometry (BD Biosciences, San Jose, CA, USA) and the data were analysed by ModFit LT Version 4.1 Software (Verity Software House, Topsham, ME, USA).
3
Cell Cycle Analysis by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Treated and untreated cells were fixed in 1% paraformaldehyde in PBS with 5mM EDTA on ice. Cells were then permeabilized in 70% ethanol for at least 30 minutes at -20°C then rehydrated in 1% BSA/0.1% Triton X-100 in PBS for 30 minutes at 4°C. Rehydrated cells were resuspended in propidium iodide (PI)/RNase staining buffer (BD pharmingen) and filtered through a 44μM pore filter. Labeled cells were analyzed on a MACS VYB flow cytometer using 535nm excitation and PI fluorescence detection at 617nm. Percentages of cells in G0/G1, S, and G2/M were calculated using ModFit LT Version 4.1.7 (Verity Software House, Topsham ME).
4
DNA Content Histograms: A Standardized Approach
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Histograms of DNA content were collected using a slightly modified method of Haase and Reed [41 (link)]. Cells were fixed in 70% ETOH and incubated in 10 mg/ml RNAse in 50 mM Tris pH 7.5 for 2 hours at 37C followed by incubation in a 5 mg/ml pepsin solution for 20 minutes at 37C. Cells were washed with 50 mM Tris pH7.5 and re-suspended in sytox green and sonicated prior to measurement of DNA content. Histograms were collected at the Flow Cytometry and Cell Sorting Shared Resource at St. Jude Children’s Research Hospital (Cancer Center Support Grant P30CA021765) on a BD Biosciences Fortessa analyzer, using 488nm excitation and detecting Sytox Green fluorescence at 510+/-10nm. Percentages of the population of cells in G0/G1, S, and G2/M as well as 2C/1C ratios were calculated using ModFit LT Version 4.1.7 (Verity Software House, Topsham ME).
5
Immunophenotyping and Cell Cycle Analysis of Cultured hPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hPSCs cultured on the aforementioned coatings were singularized with Accutase and fixed in cold 90% methanol. Primary antibodies against annexin V (1:500, Santa Cruz Biotechnology, catalog no. SC74458), Nanog (1:500, Cell Signaling Technology, catalog no. 4893), cleaved caspase-3 (1:250, Cell Signaling Technology, catalog no. 9664), Ki67 (1:500, Abcam, 15580), and an isotype control (1:500 and 1:250, mouse immunoglobulin G1 [IgG1], BD Biosciences, catalog no. 550878) were prepared in 0.3% BSA in PBS and incubated with samples overnight at 4°C. After washing, samples were incubated for 1 hr at room temperature with Alexa Fluor 488- or 568-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Life Technologies, catalog nos. A10667 and A11031, respectively) diluted 1:500 in 0.3% BSA. Samples were analyzed on a BDCanto flow cytometer using BD FACSdiva software, and negative control gating was performed on samples treated with an isotype control primary antibody and a secondary antibody identical to that used for samples. For cell-cycle analysis, samples were stained with propidium iodide (Life Technologies), and cell-cycle distribution was determined using the ModFit LT version 4 software package from Verity Software House.
6
CFSE-based T Cell Proliferation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cryopreserved PBMC were thawed and rested at 37 °C overnight in R10, then pulsed under rotation with 5 μM carboxyfluorescein succinimidyl ester (CFSE) for 10 minutes. The reaction was quenched with ice cold R10 and excess CFSE removed by washing twice with PBS + 5% FBS. PBMC were then exposed to LRA or vehicle (R10 + 0.5% DMSO) for 4 hours, washed and incubated with 2 μg/mL peptide + 20 units/mL IL-2 at 37 °C for 5 days. R10 + 0.5% DMSO + 20 units/mL was the vehicle control and 3 μg/ml PHA + 20 units/mL IL-2 was the positive control. After 5 days, the PBMC were stained with Zombie NIR viability dye, followed by CD3-PE-Dazzle 594; CD4-Brilliant Violet 650 and CD8-Brilliant Violet 510, then acquired using an LSRII flow cytometer as previously. Data were analyzed using FlowJo version 10 and ModFitLT version 4 (Verity Software House).
7
Cell Cycle Analysis of KPL-4 Xenografts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To examine the cell cycle of KPL-4 cells in vivo, KPL-4 tumor xenograft tumors were collected 4 days after the initiation of treatment and dissociated with a Tumor Dissociation kit, Human (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Then, tumor cells were isolated with a Mouse Cell Depletion kit (Miltenyi Biotec GmbH). The cell cycle of the tumor cells was examined by BD Cycletest Plus DNA Reagent kit (BD Biosciences, San Jose, CA, USA). The DNA content in each cell nucleus was determined by FACSVerse (Becton-Dickinson, Franklin Lakes, NJ, USA), and the cell cycle was analyzed by using ModFit LT Version 4 (Verity Software House, Topsham, ME, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!