I want to determine how many granulocytes infiltrate the kidneys in response to a toxin. How do I quantify them with absolute cell counts?
My method briefly: Digest kidney with collagenase & DNase, lyse RBCs, and spin over a 40/70% percoll gradient, isolate cells at interphase and stain with cd45, cd11b, and Ly6G/GR-1. I gate on CD45+ cells.
The light scatter (Fsc&Ssc) leaves a lot to be desired so I cannot determine cell number as percentage of viable/non-debris events. Any ideas on how to quantify other than cytospin?
Hello Evan,
I would use counting beads either from Spherotech (accucount) or Invitrogen (Accucheck beads, count bright) or Bangs lab (Absolute counts). These are just volumetric standards that will estimate the volume of sample analysed, from which you will deduce your cell concentration. There are the BD True Counts tubes, but they are designed to be used in a Lyse no wash method. They are certainly more accurate.
Depending on the level of accuracy you want to achieve, I would forego the Percoll phase as you may lose some cells, It would be better to include a live dead label to exclude dead cells, CD45 to identify your Granulocytes by SSC/CD45 dot plot and I would NOT wash as you can gate on live cells. The less you manipulate your samples, the more accurate your counts will be.
Also, to normalize between samples I would weigh the samples before digestion and use a cell number/per unit of mass.
Hello Evan!
Maybe something like this could help you: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Analysis/Flow-Cytometry/Flow-Cytometry-Calibration/Flow-Cytometer-Cell-Counting-Beads.html
There are some more information here: http://www.dako.com/08065_15dec05_guide_to_flow_cytometry_absolute_cell_counting_chapter17.pdf
Thank you Graciela! Do you have any direct experience with these approaches? I would be grateful for a step-wise procedure that works the first time I try it. It would be great if for once I did not have to scour the literature and then develop and troubleshoot the assay.
Unfortunately I have no experience with that, Evan! Developing cytometry protocols is really time consuming indeed... But using those beads doesn't seem very dificult, from what I've heard! If I have any new information, I will get in touch. Good luck!
Hello Evan,
I would use counting beads either from Spherotech (accucount) or Invitrogen (Accucheck beads, count bright) or Bangs lab (Absolute counts). These are just volumetric standards that will estimate the volume of sample analysed, from which you will deduce your cell concentration. There are the BD True Counts tubes, but they are designed to be used in a Lyse no wash method. They are certainly more accurate.
Depending on the level of accuracy you want to achieve, I would forego the Percoll phase as you may lose some cells, It would be better to include a live dead label to exclude dead cells, CD45 to identify your Granulocytes by SSC/CD45 dot plot and I would NOT wash as you can gate on live cells. The less you manipulate your samples, the more accurate your counts will be.
Also, to normalize between samples I would weigh the samples before digestion and use a cell number/per unit of mass.
Similar work has already been done by researchers working in renal inflammation.
For example search for Li Li, Mark D. Okusa's group in Univ. of Virginia and Dr. Jeremy Duffield in Univ. of Washington. They have published a lot. Look inside the methodology part of this paper: PMID: 17442974. Hope this helps.
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