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Should you keep blood on ice? - (Aug/19/2009 )

Hello everybody,

I isolate monocytes from mice. Because I need several animals per experiment in order to obtain enough blood, I have to store the blood until I got the blood from the last mouse. Afterwards, I continue with a histopaque protocol.
My actual question: is it OK to keep the blood on ice (in EDTA-tubes)? I was wondering if this is really a good idea because in the isolation protocol it is recommended to do the procedure on room temperature in order to avoid aggregation of monocytes. Is it sufficient to dilute the ice-cold blood in a large volume of medium at RT before starting with the histopaque procedure? Or are the cells still aggregated then (IF they aggregate while kept on ice)?
How long can you keep mouse blood at RT in a tube without damaging cells because of lack of oxygen etc.?

Thanks!
- Zorndeig

-Zorndeig-

Zorndeig on Aug 20 2009, 06:09 AM said:

Hello everybody,

I isolate monocytes from mice. Because I need several animals per experiment in order to obtain enough blood, I have to store the blood until I got the blood from the last mouse. Afterwards, I continue with a histopaque protocol.
My actual question: is it OK to keep the blood on ice (in EDTA-tubes)? I was wondering if this is really a good idea because in the isolation protocol it is recommended to do the procedure on room temperature in order to avoid aggregation of monocytes. Is it sufficient to dilute the ice-cold blood in a large volume of medium at RT before starting with the histopaque procedure? Or are the cells still aggregated then (IF they aggregate while kept on ice)?
How long can you keep mouse blood at RT in a tube without damaging cells because of lack of oxygen etc.?

Thanks!
- Zorndeig

How long does it take to collect the blood from each mouse? Can you store the blood at 4C, rather than on ice?

-swanny-

It takes approximately one hour to collect the blood from all animals. I could store the tubes in a fridge. I presume the idea of ice-cooling is to reduce metabolic activity in the blood cells so they wont get impaired by lack of oxygen (I have no idea how long the oxygen in the red blood cells is enough for the other cells in the sample) or maybe to prevent adhesion to the tube walls etc.
However, according to the histopaque protocol (Sigma-Aldrich), monocytes can aggregate at ~4C, so maybe a fridge is not better than ice.
On the other hand, the cells should endure the extensive centrifugation steps (one 30 min step and two 10 min steps), and I think the media changes between these steps do not replenish all the oxygen.

-Zorndeig-

It is not recommended to keep blood on ice as ice crystal might formed in the rbc membrane which will lyse the red blood cell. The standard working temperature should be 2-6 degree. The viability of the rbc is around 4hrs when exposed in the room temperature.

-lwumingl-

CellSpecific is correct.

We collect PBMCs from whole blood from sheep; the animal care staff collect blood during morning chores, then store the tubes in the 4C until we pick them up. We find that RBC lysis occurs during a difficult stick rather than because of storage for a few hours at 4C. To prevent aggregation of PBMCs, we collect using 1 ml 0.5 M EDTA to 10 ml whole blood, then, after transferring to a centrifuge tube, we add one volume of 1X PBS + 2mM EDTA.

regards,

lab rat

-lab rat-