Comparison of Clonogenic Survival Data Obtained by Pre- and Post-Irradiation Methods

Clonogenic assays are the gold standard to measure in vitro radiosensitivity, which use two cell plating methods, before or after irradiation (IR). However, the effect of the plating method on the experimental outcome remains unelucidated. By using common cancer cell lines, here we demonstrate that pre-IR and post-IR plating methods have a negligible effect on the clonogenic assay-derived photon sensitivity as assessed by SF2, SF4, SF6, SF8, D10, or D50 (N.B. SFx indicates the survival at X Gy; Dx indicates the dose providing X% survival). These data provide important biological insight that supports inter-study comparison and integrated analysis of published clonogenic assay data regardless of the plating method used.

Culture of IMR90 Cells

This protocol describes the thawing, culturing, and cryopreservation of the human lung fibroblast cell line IMR90. The attached methods document is a formal version of the information included here. The attached worksheet is a fillable PDF that can be used to maintain cell passage records using this protocol. Please note: Deviation from the three-day passage cycle and cell plating density described here typically results in greater culture and experimental variability.Disclaimer: The contents of this article have been reviewed by the US Environmental Protection Agency and approved for publication and do not necessarily represent Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.

Culture of 16HBE14o- Cells

This protocol describes the thawing, culturing, and cryopreservation of the human bronchial epithelial cell line 16HBE14o- (referred to as “16HBE”). The attached methods document is a formal version of the information included here. The attached worksheet is a fillable PDF that can be used to maintain cell passage records using this protocol.Please note: Deviation from the three-day passage cycle and cell plating density described here typically results in greater culture and experimental variability.Disclaimer: The contents of this article have been reviewed by the US Environmental Protection Agency and approved for publication and do not necessarily represent Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.

The effectiveness of glass beads for plating cell cultures

Cell plating, the spreading out of a liquid suspension of cells on a surface followed by colony growth, is a common laboratory procedure in microbiology. Despite this, the exact impact of its parameters on colony growth has not been extensively studied. A common protocol involves the shaking of glass beads within a petri dish containing solid growth media. We investigated the effects of multiple parameters in this protocol - the number of beads, the shape of movement, and the number of movements. Standard suspensions ofEscherichia coliwere spread while varying these parameters to assess their impact on colony growth. Results were compared by a number of metrics - the number of colonies, the mean distance between closest colonies, and the variability and uniformity of their spatial distribution. Finally, we devised a mathematical model of shifting billiard to explain the heterogeneities in the observed spatial patterns. Exploring the parameters that affect the most fundamental techniques in microbiology allows us to better understand their function, giving us the ability to precisely control their outputs for our exact needs.

Differential Expression of Tyrosine Hydroxylase Protein and Apoptosis-Related Genes in Differentiated and Undifferentiated SH-SY5Y Neuroblastoma Cells Treated with MPP+

The human neuroblastoma SH-SY5Y cell line has been used as a dopaminergic cell model for Parkinson’s disease research. Whether undifferentiated or differentiated SH-SY5Y cells are more suitable remains controversial. This study aims to evaluate the expression of apoptosis-related mRNAs activated by MPP+and evaluate the differential expression of tyrosine hydroxylase (TH) in undifferentiated and retinoic acid- (RA-) induced differentiated cells. The western blot results showed a gradual decrease in TH in undifferentiated cells and a gradual increase in TH in differentiated cells from days 4 to 10 after cell plating. Immunostaining revealed a gradual increase in TH along with neuritic outgrowth in differentiated cells on days 4 and 7 of RA treatment. For the study on cell susceptibility to MPP+and the expression of apoptosis-related genes, MTT assay showed a decrease in cell viability to approximately 50% requiring 500 and 1000 μM of MPP+for undifferentiated and RA-differentiated cells, respectively. Using real-time RT-PCR, treatment with 500 μM MPP+led to significant increases in the Bax/Bcl-2 ratio, p53, and caspase-3 in undifferentiated cells but was without significance in differentiated cells. In conclusion, differentiated cells may be more suitable, and the shorter duration of RA differentiation may make the SH-SY5Y cell model more accessible.