Hi,
I am using Absolute counting beads to count the number of neutrophils in mouse peripheral blood. I was wondering if somebody has done similar work. I am using Count Bright beads from ThermoFisher.
i am collecting 50ul of blood ( collected in EDTA coated tubes), staining with 1A8 and CD45 antibody for 15-20 min and then using ammonium chloride lysis buffer to get rid of rbc's. I am not centrifuging the samples, to avoid loosing the cells. After the lysis I read the samples on FACS Canto using FACS Diva6 software. All further aanalysis is performed using FlowJo.
currently, I am concerned with two issues:
1. Debri from rbc lysis. Since I am not centrifuging the samples, I am getting a lot of debri from rbc lysis which is far more into the right shoulder of FSC than I expected it, so it kind of complies over the leukocyte population.
I am actually concerned if in fact all the rbcs are lysing? I prepared fresh lysis buffer per no-wash rbc lysis, but I am seeing the same results
I have to collect a lot of events to get a decent leukocyte population. To avoid the debri, I started gating on the main leukocyte population and recording only those events during acquisition. I am concerned I may be loosing some cells that fall outside of the gate for the majority of leukocyte population. I was wondering if somebody can suggest a strategy to address that.
2. The size of the beads
the beads I am currently using are 7um which should be the relative size of the leukocyte population. I expected them to appear close on the plot. But in fact, i have to reduce SSC largely to fit the beads on the plot and that really compresses my leukocyte population, which I am not sure if I should worry about yet. I vortex the beads a lot to avoid clumping but the problem seems to persist.
Any suggestions would be helpful
i will include pictures as soon as
thank you
Hi Alex,
I have not done the absolute counting of neutrophils using beads so can't say a lot about it. However, I do comparative quantification of neutrophils in mouse peripheral blood with different treatments.
What is the concentration of your lysis buffer? How long do you lyse in ammonium chloride lysis buffer? Your cell quality and quantity as well as the efficiency of lysis depends on it.
In my hands, I found ammonium chloride to be not a good lysis buffer for neutrophil isolation or quantification. The reason being same as you mentioned a) not enough lysis if the incubation is short b) and getting lots of cells lysed (leukocytes especially monocytes and granulocyte populations) c) I believe most of granulocytes get activated with ammonium chloride treatment as I used to see my neutrophils scattered rather than getting a nice tight population as I do now. Hence I switched to BD FACS lysis buffer for lysing RBCs and it resolved all the issues.
Do you need the cells to be sorted live? If you do then you can't use BD FACS lysis buffer as it fixes cells while lysing RBCs.
Good luck with your experiment.
Jyoti
To get rid of debris, increase the threshold value of acquisition.
For not compressing your lymphocytes data, you can increase the voltage. Generally beads will not overlap with lymphocytes but the beads will appear at right corner as small cluster. Mix the bead properly to avoid lumps and and you can record smaller amount of data if its too crowded.
Hi your final volume should match the final volume of the counting beads and I suggest you pick up the beads in another channel so as not to affect your neutrophil discrimination. They will appear bright on many channels.. try FL4 and plot SS against time.
Hi Aleks,
Change your threshold setting. I would suggest using 'OR' with the CD45 channel and an unrelated channel where your beads fluoresce. That should push your rbc's and debris below threshold, giving you cleaner and smaller data files. Remember to keep the flow rate set to 1 to keep the core stream tight. You can also use a live gate to eliminate unwanted debris from your final fcs files. Just be very careful as events not included in that gate will not be saved in the file.
Don't decrease SSC. The beads are there, just slammed into the topmost channel. To gate on them, use a histogram in one of the unrelated fluorescent bead channels, such as the channel used to set the threshold. It's not unusual to see beads super high or super low on a scatter plot. As much as we say FSC is size and SSC is granularity, that really isn't true. There are a host of other things affecting those two signals.
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