Using ACK at what temperature for how long? - (Oct/17/2007 )
Hello friends,
In the past, I have received many suggestions of how to use ACK to lyse rbc and none of them worked. Some have stated to use this buffer at 4C for 10 minutes and others have said to use this buffer at 37C for 5 minutes. You never use ACK at either 37C or at 4C for only 10 minutes.
DOES ANYONE USE ACK AT ROOM TEMPERATURE BETWEEN 5-10 MINUTES?
Please tell me exactly how many minutes and what the cell suspension solution is composed of (what is the buffer) before adding this buffer. This will help me tweak the procedure. 
Hi there!
I resuspended the cell pellet in ACK, so there's no residual buffer to start with. Cell are incubated at Rt for 5', and gently mixed every other minute, then centrifuged at 300g for another 5', and subsequently washed 3 times with either PBS or RPMI 1640... You should see that the pellet gets whiter with every wash...Worked for me....
Mike
I resuspended the cell pellet in ACK, so there's no residual buffer to start with. Cell are incubated at Rt for 5', and gently mixed every other minute, then centrifuged at 300g for another 5', and subsequently washed 3 times with either PBS or RPMI 1640... You should see that the pellet gets whiter with every wash...Worked for me....
Mike
Hi,
WHAT WERE THE CELLS ORIGINALLY SUSPENDED IN BEFORE THE ACK? You should resuspend these cells first in a buffer BEFORE adding the ACK because of cell waste products in contact with these cells.
My protocol is similar. With mouse spleen/lymph node cells, I resuspend the cells in 5 ml Ack buffer, incubate at room temp for 5 min with occasional mixing, add 10 ml IMDM media with FBS, pellet the cells and wash once with more IMDM. Works fine for me.
After spinning down the cells, I resuspend them in 3ml-5ml of ACK at room temperature and mix them well and keep for not more than 3 mins occasionally mixing by rocking the tube gently. I then fill up with medium/PBS and spin it down and resuspend the cells in medium. The pellet becomes white.
I was taught that it should not give problem increasing the volume of ACK used but should not keep it longer as white cells get clumped. It works fine with spleen and thymocytes of mice but when I attempt to lyse RBCs in whole blood, I still get residual RBCs at the end of 3 mins.
I have seen some protocols recommending ice cold ACK. Can someone tell the difference we get when using cold ACK and RT ACK.