Hi Hyde if the cells get too much radiation they will basically die from apoptosis.

Perhaps you could try to "titre" the radiation dose - either by time (in one flask 500 rad for 5 min, next flask 10 min next flask 20 min etc) or by dose (fixed time with500 rad, 600 rad, 700 rad etc).

Hopefully one set of parameters will be ideal for you - does the paper you are trying to replicate mention the duration of the ionising radiation or the confluence of the cells at the time they received the dose?

Also are the fibroblasts you are looking at primary dermal fibroblasts or an immortalised cell line? Primary cells will naturally be more sensitive to radiation. I would check that you are using the same type/source of fibroblasts that they do in the paper.

Hope this helps,

Gary

Hi Hyde - if the cells receive too much radiation they will die (through apoptosis).

Are you using immortalised fibroblasts (such as a cell line) or are the cells primary cells?

Primary cells will probably be more sensitive to radiation. I would check that you are using the cells that were used in the original paper.

I would also check at what confluence the cells are at when exposed to radiation. From personal experience fibroblasts do not really suffer from density-dependence but this may become more critical under the affects of ionising radiation.

If you are performing the experiment the same as they do in the paper (same cell-type, same confluence, media etc) you might want to try varying either the dose at a fixed time (500 rad 10 min, 600 rad 10 min etc) or the dose itself (500 rad 10 min, 600 rad 10 min etc) until you get an effect.

Sorry I can't be more help,

Gary

sorry about the 2x answer! Thought I had "lost" the first one.... :)

I want to "titre" the radiation dose， however there are some problems.

1,The machine is work x rad per min and after working I must wait 15 mins before get the cell out from the machine. The cell will wait long time. And the machine is in a public platform which is away from my lab.  How about carry cell with ice box？

2,I digest cell from the dish as we do with the mice feeder. What is the different, suspension or adherent？ 

The cells are best transported at room temperature (not ice - this will give a hypothermic shock to the cells). Fibroblasts will be okay at room temperature for about an hour or so if they are in buffered media but you should also take your "control" cells along too for the ride so that they are subject to the same environment/temperature (you want to measure effects of radiation, not radiation + temperature shock).

There are two types of cell in general - suspension and adherent cells. Suspension cells are mostly types of blood cells with all other types being adherent.

Adherent cells like to be attached to something (as they are attached in the body to the extra-cellular matrix which contains proteins such as Fibronectin and collagen that the cells "cling" to).

When adherent cells are detached from the dish that they grow in unless they re-attach in an hour or so they begin to die (through a process called Anoikis, a form of cell suicide).

As mentioned adherent cells like to be "stuck down", many adherent cells also don't like to be lonely - they like the presence of other cells nearby (in other words they can be "density dependent"). Thus how many cells are covering your dish (the "% confluence") can sometimes also affect the results of your experiments.

Fibroblasts are not particularly density dependent - they do not mind being alone. However if you are irradiating them when they are 100% confluent (fully covering the dish with no gaps between the cells) you may find it harder to kill them with radiation. When cells reach confluence they often stop growing, the cells stop cycling and tend to enter the G0/G1 phase of the cell cycle (i.e. they "switch off" DNA replication etc).

This is why I was saying that you should perform the experiment on cells at the same density that they did in the paper you are trying to replicate (if it is mentioned - if it is not mentioned assume that the cells are just sub-confluent which is ~ 90% confluence as people tend to mention if the cell density is low or if the cells are confluent.).

I hope this makes sense?

Gary

Hello Hyde,

We did this with 3T3 fibroblasts. We irradiated the cells attached to the plate at approx. 80% confluence. We kept them in culture medium during irradiation. Our best irradiation dose for subletal damage to stop proliferation was 80Gy. The cells stayed alive for 3 weeks after that, but with no more proliferation. We used an MTT test to evaluate cell viability. We also had to transport the cells to another facility, and our X-ray machine was very low intensity, so the irradiation took around 30 min. 

Good luck!

thanks so much. Tomorrow i will try it.