We have some frozen blood in -20 freezer. They are there for about 1-2 months. Now I need to isolate WBCs. Does anyone know of an experiment I can use? I need them for DNA extraction.
you can use the whole blood kit for DNA extraction it will help you
Yes of course, I've done this several times and I was satisfied with the results of DNA extraction.
thank you Merin and Mostafa. Yes, I know I can isolate DNA from that frozen blood. But I'm working on a protocol which needs isolated WBCs, and I was just curious if anyone know of an experiment I can use to isolate buffy coat from frozen blood.
Dear Javad, I believe that the freeze-thaw cycle will simply induce major destruction of all blood cells: red blood cells, white blood cells and platelets. As such you will not be able to isolate intact WBCs and I am not even sure that you will get a well defined buffy coat layer, as the cell debris/stroma may not sediment as expected. In addition, due to the hemolysis, the plasma will be highly contaminated by hemoglobin. You should really start from freshly collected blood in the presence of anticoagulant and centrifuge it at the right g force and time to get a correct buffy coat. Best, Thierry
Thank you very much Thierry. Yes you're right, I think the plasma would be dark red. But I'm still curious and like to give it a try. Do you think I need to use a higher or lower g force?
Dear Javad, personnally, I would tend to use a higher g force since you need to sediment cell debris rather than intact cells. Best, Thierry
Dear Thierry, actually I tried it, and all I saw was nothing, I even didn't see any fraction. It looks impossible to isolate buffy coat from frozen blood with a normal protocol.
Dear Javad, yes, I am not too surprised. probably all cell debris sedimented at the buttom, and the hemoglobin and plasma mixed together. Thierry
Well, even if it appears almost utopic, could it be feasable to use a gel filtration on some kind of a "sephadex" colulmn ? seems stupid but, you know... sometimes if you unfreez very slowly, gently, with -say - lots of glycerol... and everybody knows taht only one freez-thawing cycle is not enough to lyse cells..
Dear Gérard Mauco, yes it maybe possible, but actually I'm seeking for a cheap method and also this method doesn't seem practical.
The "method" should be cheap but to be honest : forget it, there is probably no means to isolate functional WBC from frozen blood if nothing has been done before hand to preserve cell integrity....and if any possible the cells would be so damaged and epitopes so modified that, to be quite rude, I do not see any interest of doing it.... good luck !
Yes, It is possible. The frozen blood can be allowed to thaw. Once thawed the blood sample can be centrifuged at 3000 rpm for 20 minutes. The buffy coat can be found as a buffy or whitish thin layer at the intersection of plasma and red cells. However, the freeze thaw cycle can damage the white cells and also the red cells. The buffy coat may not appear buffy but somewhat reddish. Using the buffy coat DNA may be extracted.
HI.
Yes its very hard to isolate WBC from frozen buffy coats. However you can still get DNA from frozen whole blood without WBC separation. Try a method such as salting out (Miller et al. 1989) or phenol chloroform.
Firstly, I wonder if you added any freezing protecting agent, such as "glycerin" before you froze the blood. If you did, then after thawing and washing with physiological saline, you are able to keep the integrity of the blood. Secondly, I wonder if you could use the blood filter, such as Pal filter to collect the WBCs after washing. After all, this is how the WBCs were removed before the RBCs are transfused into patient's body at the patient's bed site. Then, you basically can wash out the collected WBCs from the pal filter and centrifuge it down to collect the WBCs. Hope that it will work it out for you.
Impossible task since cells will likely rupture with the freeze-thaw cycle (damage done by ice crystals), unless you had added cryo-preservation agents before freezing.
Man-Chiu Poon
Most of the cells will lyse within buffy coat preparation procedure. Why don't you use simply ACK lysis buffer to isolate leucocytes ?
Dear Javad,
If your goal is to extract DNA from the buffy coat fraction, despite the fresh blood you should collect the lower fraction in frozen blood after centrifugation. However, it is not visible. But if you are planning to seperate intact WBC, I am regret to say that its almost impossible without using protectors before freezing.
In the end, is there a good protocol for obtain leukocytes from frozen blood? I know that I can obtain the DNA, but is it possible to obtain leukocytes for WB experiments?
Dear everyone..
Sometimes I have to delay to isolate the buffy coat after drawing the blood for 6 - 8 hrs. I'm confused which one is recommended: 1. Should I centrifuge it first after drawing, keep it in refrig (or in room temperature?)then do the isolation 6-8 hrs later; or 2. Should I leave it in refrig (or room temperrature?) then after 6-8 hrs do the centrifugation and isolation.
Thank you
Regards, Deni
- Badges
- Science method