Im looking at the leukocyte in mouse peripheral blood. Lysing rbcs with ack
having some issues with incubation time
Tried 10-15min
Figure 1
the plot looks different from previous trials ( figure not included but resembles more figure 2) and from publications
i was concerned if the main population could be unlysed rbc cells...
so in two separate tubes, I increased incubation to 30min
figure 2 and 3( don't pay attention to the blue population on figure 3)
one tube gives figure 2, another tube - figure 3
???
I'm confused, can somebody help with interpreting results?
Apologies, the number of events collected is not the same between tubes which goes into
...
the flow rate of events/ sec
tube 1( figure 1) and tube 3 ( figure 3): flow rate 200- 500events/sec
Tube 2: the flow rate was at least 10fold higher...
why such a big difference?
Could it be from lysing more cells and picking up a higher rate of debri events?
Apologies for the absence of better pictures the better pictures.
You shouldn't incubate your samples with ACK solution for more than 5 minutes. Prolonged incubation damages the WBC populations so their SSC and FSC properties change. If the RBC lysis is incomplete, you could do another round of 5 minutes incubation with FRESH ACK. 50-100ul blood sample is enough for flow cytometry. Also, you can gate out the RBC from WBC during analysis if the lysis is incomplete.
Regarding your figures:
Your WBC populations appear after 50K for FSC-A. As you can see, the longer you incubated the samples, more cells shifted to below 50K which were usually dead cells or debris...
Figure 1 differs from Figure 2 & 3 mainly due to difference ACK treatment time. Figure 2 differs from Figure 3 mainly due to difference in number of events displayed on the dot plot. Surly your software will allow to display comparable cells (dots) on the plot than you can see that figure 2 & 3 are similar.
regarding the better way of the study, please avoid over lysis. Due to very long time of lysis, figure 2 and 3 shows high amount of debris.
You need to optimize the number of cells to be taken per tube and the ACK treatment. Do not treat upto 15 mins or so. From figure 1 it appears that you are very close to get good results.
Dont over lyse, avoid temperature fluctuation, do not entangle your work unnecessary (as cells will be healthier if you start acquiring the data as early as possible from the time of taking out cells from the experimental model).
Thank you Kai Ling and Mohammad for your response. I am sorry it has taken me a while to get back to you
I certainly agree with both of you. I was unsure if my interpretation was correct, so I posted the question to get some feedback. I feel more comfortable now moving forward. Thank you
Dear Aleks
I am working also to isolate mice wbc. I had problems to lyse the RBC. would you please tell me which lysing buffer are you using? and whats the amount of blood? Thank you
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