The largest database of trusted experimental protocols

3 protocols using actinomycin d

1

Metaphase Preparation and FISH Chromosome Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Metaphase prep for cell culture was modified from the preparation of metaphase chromosomes of adherent cells (NCI National Institute of Health, 2006 ). All incubations were for 15 mins and all centrifugations were performed for 8 mins at 350xg. First, 300μl of Actinomycin D (AG Scientific, San Diego, CA) at 100μg/mL was added to the flask and incubated for 15mins. Next, 300μl KaryoMax® Colecmid® Solution (Gibco® Life Technologies, Grand Island, NY) was added to the flask. FISH protocols were adapted from Coriell. The centromeric probe used was CEP 11 (D11Z1) SpectrumGreen probe 11p11.11-q11 Alpha Satellite DNA (06J37-021 Abbott Molecular, Abbot Park, IL). In addition, the slides were stained with ProLong gold antifade reagent with DAPI (P36935 Molecular Probes Life Technologies Grand Island, NY) and stored at −20°C. Images were taken at 63x on a Leica CTR microscope using Perkin Elmer Volocity 6.1.1.
+ Open protocol
+ Expand
2

Serum and Leucine Starvation Response

Check if the same lab product or an alternative is used in the 5 most similar protocols
HeLa cells (ATCC, Manassas, VA, Cat. # CCL-2) were maintained in culture at 37°C in a humidified incubator with 5% CO2 in high glucose Dulbecco's modified Eagle's Medium (DMEM, Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (Atlas Biologicals, Fort Collins, CO) and 1% penicillin-streptomycin (Invitrogen). On the day of the experiment, cells were placed in serum-free medium (DMEM) or leucine-free medium (Atlanta Biologicals, Lawrenceville, GA) for 10 hours. After 10 hours of starvation, 10% serum or 0.76 mM leucine (Sigma, St. Louis, MO) was re-introduced into the culture medium for the indicated times. For experiments using Actinomycin D (Sigma) or cycloheximide (A.G. Scientific, San Diego, CA), cells were prepared as described above with the exception that either Actinomycin D (2ug/mL) or cycloheximide (100ug/mL) was added to the medium for 15 min or 2 min, respectively, prior to the re-introduction of serum or leucine.
+ Open protocol
+ Expand
3

Serum and Leucine Starvation of HeLa Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
HeLa cells (ATCC, Manassas, VA, Cat. # CCL-2) were maintained in culture at 37 °C in a humidified incubator with 5% CO2 in high glucose Dulbecco's modified Eagle's Medium (DMEM, Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (Atlas Biologicals, Fort Collins, CO) and 1% penicillin-streptomycin (Invitrogen). On the day of the experiment, cells were placed in serum-free medium (DMEM) or leucine-free medium (Atlanta Biologicals, Lawrenceville, GA) for 10 h. After 10 h of starvation, 10% serum or 0.76 mM leucine (Sigma, St. Louis, MO) was re-introduced into the culture medium for the indicated times. For experiments using Actinomycin D (Sigma) or cycloheximide (A.G. Scientific, San Diego, CA), cells were prepared as described above with the exception that either Actinomycin D (2 μg/mL) or cycloheximide (100 μg/mL) was added to the medium for 15 min or 2 min, respectively, prior to the re-introduction of serum or leucine.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!