Post Wed Apr 06, 2016 8:12 pm

Staining aged (shipped) blood for CyTOF

We are interested in running a 5 day time course from patients enrolled in a clinical trial by CyTOF. I was wondering if anyone has any experience on running CyTOF on shipped blood specimens.

The general workflow that we were considering was:

1. PBMC preparation by ficoll gradient, then wash twice
2. Stain with cis-platin for live dead, then wash twice
3. Surface stain with metal-conjugated antibodies and fluorescent labelled antiobodies, then wash twice
4. Secondary stain with anti-fluorescent metal-conjugated secondary antibodies, then wash twice
5. Fix with Foxp3 fix/perm at room temperature for 30 minutes, then wash twice with perm buffer
6. Stain with intracellular antibodies, then wash twice with staining buffer
7. Resuspend in Foxp3 fix/perm with iridium, and incubate overnight at 4*C
8. Wash out iridium with staining buffer, then wash twice in milliQ water
9. Resuspend in distilled water with elemental bead solution

The above has worked great on fresh PBMCs. However, we found that the older the blood was, there was more iridium streaking. The iridium streaking was eliminated by using a dead cell removal kit, but that also removed some of our populations of interest.

We changed to shipping in refrigerated boxes. We also are interested in just lysing whole blood instead of doing a ficoll preparation. The granulocytes may also be very interesting with what we are studying and will also end up in the PBMC layer in aged blood. We would love to include the granulocytes in our analysis if possible.

We are using a WBC syringe filter to remove most of the erythrocytes and platelets first before lysis. Unfortunately, neither has solved our iridium streaking problem on aged blood.

We have not considered doing a hard PFA fix just before washing into water. The cells are fixed in two separate steps, so we think that the cells should be sufficiently fixed. The cells obtained from our shipped samples work great by flow cytometry.

Has anyone else successfully run aged blood on CyTOF?
Attachments
Aged blood CyTOF staining.pptx
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