Clonogenic assay of cells in vitro

Abstract

Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony is defined to consist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo "unlimited" division. Clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. Only a fraction of seeded cells retains the capacity to produce colonies. Before or after treatment, cells are seeded out in appropriate dilutions to form colonies in 1-3 weeks. Colonies are fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using a stereomicroscope. A method for the analysis of radiation dose-survival curves is included.

Targeting the Autophagy Pathway Using Ectopic Expression of Beclin 1 in Combination with Rapamycin in Drug-Resistant v-Ha-ras-Transformed NIH 3T3 Cells

“…4F). Clonogenic assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony and can also be used to determine the effectiveness of other cytotoxic agents (Franken et al, 2006). When used as a single agent, rapamycin inhibited Ras-NIH 3T3/Mdr cell clonogenic survival.…”

An Apoferritin‐based Drug Delivery System for the Tyrosine Kinase Inhibitor Gefitinib

“…Clonogenic assays were performed to determine whether individual SKBR3 cells were able to survive challenge with Gefi tinib or H-AFt-encapsulated-Gefi tinib and subsequently form progeny colonies, indicative of tumor repopulation. [ 37 ] After 24 h exposure to agents followed by 13 d incubation with medium alone, H-AFt-encapsulated-Gefi tinib demonstrated lower potency than Gefi tinib ( Figure 4 ). The survival fraction (SF) of cells treated with Gefi tinib was 50.7% ± 1.5% (1 × 10 −6 M ) and 3.5% ± 1.0% (5 × 10 −6 M ) compared to control whereas SF of cells treated with H-AFt-encapsulated-Gefi tinib was 71.8% ± 0.5% (1 × 10 −6 M ) and 34.4% ± 5.2% (5 × 10 −6 M ).…”

“…SF were calculated: SF = Plating effi ciency of treated sample/Plating effi ciency of control × 100%. [ 37 ] Release of Gefi tinib from H-AFt : Dialysis bags (cut-off 8 kDa) containing H-AFt-encapsulated-Gefi tinib (100 × 10 −6 M ) and Gefi tinib (100 × 10 −6 M ) diluted in 20 × 10 −3 M Tris buffer (900 µL) were placed in separate beakers containing 20 × 10 −3 M Tris buffer at pH 2, 4 or 7.5 at 37 °C, 5% CO 2 . Released Gefi tinib was quantifi ed after 2, 6, 12, and 24 h by UV spectrophotometry at 250 nm.…”

Inhibition of insulin‐like growth factor 1 receptor enhances the efficacy of sorafenib in inhibiting hepatocellular carcinoma cell growth and survival

“…Hep3B cells were seeded into 12‐well plates (6 × 10 3 cells/well) for 24 hours before treatment as described 21. After 48 hours of treatment with dimethyl sulfoxide, 1.25 μM sorafenib, 0.5 μM ceritinib, or a combination of both drugs, media was changed and the cells were cultured for 14 days.…”