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How to induce early apoptosis in cells without using chemicals or UV

cell biology cell culture immunology cell death apoptosis

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#1 CMIRC

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Posted 15 August 2012 - 09:53 AM

I am trying to induce early apoptosis in PC3m cells and K562 cells using heat induction. However I have been unsuccessful. I either get viable cells or dead cells and nothing in between. My method in brief is suspending 250 000 - 500 000 cells per eppendorf microfuge tube in 100 ul of RPMI. I then incubate the cells in a water bath preheated at 43 degrees celcius for 40, 30, 15, 10 mins. After incubation I check for it using Viability reagent using the Flow cytometer. However with both the cell lines I either get high viability or high cell death.

I would be greatly appreciated if somebody can offer me some advice on how to achieve early apoptosis or even late apoptosis. A protocol that has been tried and tested would be good, however I am open to all suggestions.

Hope to hear from someone soon

CMIRC

#2 Curtis

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Posted 15 August 2012 - 09:11 PM

I'm not familiar with this method of heating up cells. where did you get this? what is the purpose of this method? I normally use UV to induce intrinsic pathway of apoptosis or use staurosporine or infect with a virus.

You said you don't want to use chemicals or reagents. But what about CD95 mAb (FAS-L antibody)? Can't you even use that?

http://www.abcam.com...e&rid=11368

what are your viability reagents? if you are using PI you must run in flowcytometer quickly after addition.

#3 CMIRC

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Posted 16 August 2012 - 03:59 AM

Thank you for the reply it is very informative.

This method is not common but has been published by several journals (google heat induced apoptosis). However the methodology is not explained in detail. The purpose of this method is to avoid any chemical intervention that would cause apoptosis and UV has a possibility of mutating the DNA and also I do not have the means of using UV in a more controlled manner to induce apoptosis. I have used chemical means in the past and they work brilliantly however my project requirement is to use heat.

The use of CD95 mAb sounds good. I will try to implement that as one of the treatments. However I still need to use hyperthermia as a method of inducing apoptosis.

I use the Guava (Millipore) Nexin reagent that has anexin 5 which binds to PS that is expressed on cells undergoing apoptosis. And I follow the instructions as per the manufacturer. However I never get early or late apoptosis its always either dead or viable cells. Hence I would appreciate any help from anyone who has experience before or current on this topic.

Also Mettaler Scientist you mentioned you have used UV before. Could you please elaborate as to which cells and how long was the exposure and also did you get early or late apoptosis?

Hope to hear form you soon or anyone

CMIRC

#4 Curtis

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Posted 18 August 2012 - 07:21 PM

in most articles people give energy value for UV exposure. They put their flasks or dishes in crosslinker machine under 300uJ/cm2 (you have to check this yourself, it's long time I haven't done this).

But I didn't have crosslinker or UV meter to measure the energy. So I just grew my cells in 10cm2 dish and then put the dish in laminarflow and switched on the UV lamp. I put all my sample almost 20cm below the lamp and kept them there for 5-45 minutes. After 45 minutes membrane blebbing was apparent but I don't recommend it. I think it's better to expose them to UV only for 5 minutes then put them back in the incubator, they will eventually die. I feel like 45 minutes exposure will boil the cells. But you can try this...keep your cells under UV and check them under microscope at different time points.

Remember if you grow them in dish you have to remove the lid before exposure because UV cannot pass through the polymer.

#5 madelingirly

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Posted 02 September 2012 - 10:34 PM

Could u check this method

A good positive control for apoptosis: Heat shock the cells. Incubate 1 minute @ 56°C followed by incubation @ 37°C
dependent on cell type. Usually 1 hour @ 37°C after shock is sufficient to induce 50% apoptosis for most cell lines

#6 CMIRC

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Posted 08 October 2012 - 04:51 PM

Could u check this method

A good positive control for apoptosis: Heat shock the cells. Incubate 1 minute @ 56°C followed by incubation @ 37°C
dependent on cell type. Usually 1 hour @ 37°C after shock is sufficient to induce 50% apoptosis for most cell lines


Thank you Very much for proposing this method. I tried it on K562 and HL60 cells as well as PC3M and I get apoptosis. I tried both 56°C and 60°C and 37°C incubation thereafter and 56°C gave me 50% apoptosis out of which about 40-45 % was in late apoptosis and remarkably 60°C gave me 90% apoptosis (late apoptosis). This is an amazing method. Thank you for proposing it. Thanks once again.





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