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PLease help


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5 replies to this topic

#1 vraev

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Posted 28 October 2009 - 10:50 PM

Hi guys,

In haste as I was about to finish a long day of experiments (19hr day of continous work) by running some splenocytes on a cytometer, I forgot my box of antibodies on the table at room temperature for 3 hrs. The box is opaque so they are not exposed to light.

I use colors such as

FITC
APC
APC-Cy7
PE
PE-Cy7
DAPI
pacific blue

Most of these antibodes are brand new. I came back after a 3h of flow session and realized my mistake and put them back immediately at 4C in the fridge. Although, now I am freaked out if the antibodies have degraded.

Can someone tell me if all hope is lost ? WIll those conjugated dyes degrade within hours at room temp? It was a maximum of 3h. I remember reading an old publication that most normal flourochromes such as FITC are stable at room temp for like 700h or so before the loss of signal.

I am dying with dread if those Ab have degraded. I worked hard to standardize that panel and my supervisor won't be very happy to hear if they degraded due to such a stupid error.

thanks,

V

#2 elpollodiablo

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Posted 29 October 2009 - 01:36 AM

They should be just fine, don't worry about it

#3 vraev

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Posted 29 October 2009 - 07:43 AM

But what about the conjugates like APC-Cy7 and PE-Cy7?? ;)

#4 elpollodiablo

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Posted 29 October 2009 - 12:12 PM

But what about the conjugates like APC-Cy7 and PE-Cy7?? ;)


My guess would be that as long as they haven't been exposed to light for prolonged periods of time or they've been heated to way above RT, you are in the clear. I've been using PE and FITC conjugated ABs that were 4 years too old, but still worked fine. I find it very hard to believe that 3 hours at RT would inactivate conjugates. Try and use them, and see what happens. I'm confident you'll find that they are just fine.

#5 miBunny

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Posted 29 October 2009 - 06:04 PM

This happens all the time to us, antibody boxes will be left out for 1 to 2 hours (PI is the worst offender) and we have never seen any degradation with any of our fluorochromes (tandem dyes included). Considering how often this happens to us, our boxes of antibodies have had hours at room temp. You should be fine :)

#6 vraev

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Posted 29 October 2009 - 06:05 PM

thanks!! The reason for the concern is that APC-Cy7 and PE-CY7 are especially notorious for splitting back to APC and PE causing spillover into the other channels. The former has been known to be especially unstable to fixing buffer, temperature variations, light exposure etc.

So yeah...thanks a lot for helping. I will maybe check it with fresh splenocytes and see if there is any spillover.

thanks for putting my mind at ease for now though. lol!




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