Hi all

I am trying to investigate the role of my gene i.e N-WASP in skin carcinogenesis. For this, I am doing Focus Formation assay in NIH3t3 cells.

Protocol: (Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene Angel Alvarez , Gustavo A. Barisone , Elva Diaz, 2014).

I am currently trying to optimize this assay in NIH3t3 cells and facing some challenges. 

Background

I am performing two stage carcinogenesis.

Tumor Initiator: Mutated ras

Tumor Promoter: N-WASP knockdown

First, I infect NIH3t3 cells with mutated ras retrovirus, select them with antibiotic (G418, Geneticin in my case). Afterwards, I infect these ras stable cells with another retrovirus having shRNA for my gene of interest.

In second case I do not select, but allow them to grow over for a period of two weeks for Foci to form and compare with control.

1. Ras + Knockdown --> Test

2. Ras + No Knockdown--> Control

3. Ras (only Ras stable cells and no second Infection)--> Control

Problem

After a period of two weeks, the cells come off from the dish, making it difficult to count foci in entire dish. I have attached some pictures here.

If anyone could share their suggestions or any protocols that could help will be greatly appreciated. 

Thanks & Regards

Apoorva Verma