Additional Q&A with Dr. Adrian Kendal
1. What is the amount of antibodies added for CITE-seq in comparison with flow cytometry? Thanks.
I used 96U plates, up to 50,000 cells per well. After washing cells, they were incubated at 4° C for 30 mins with 10 μl of BioLegend stock concentration human Fc blocking buffer. I then added equivalent of 0.5 μg to each well of surface TotalSeq™ mAb (stock concentration is 0.5 mg/ml) and then appropriate hash mAb. I followed this protocol: https://citeseq.files.wordpress.com/2019/02/cite-seq_and_hashing_protocol_190213.pdf I suspect that the concentration could be reduced in future to match whatever has previously worked for the same fluorophore-conjugated mAb on your cells. The difficulty with tendon fibroblasts is that they autofluoresce to a high extent. I suspect that over time, we will be able to dilute the TotalSeq™-A mAb, but this was/is a low priority because the antibody costs are much lower than the cost of obtaining tissue and sequencing the cells. I did see some cells with >1 hash antibody bound to the surface, so will reduce the concentration of hash/labelling mAb in future. I think that basically, if you have a good mAb with a good binding track record for your cells, then go with that concentration - conjugating it with oligonucleotide has little impact. BioLegend's QC work says as much.
2. Nice talk. Can you tell us what is the average efficiency of your labelling for Citeseq and hashing? and what were the conditions you used?
See my answer to question #1 for conditions used. I cannot tell you about average efficiency of labelling. This is something that can be checked using a fluoro-conjugated mAb of same mAb and flow cytometry.
3. Were you able to see an effect of the age of your patients within your healthy or diseased groups?
Thank you. Not with this limited dataset. I imagine there will be age-related effects on tendon cell subtype composition, function and differential gene expression. One of the associated issues then becomes what we define as "normal control" tissue - should it be healthy young tendon or age-matched asymptomatic tendon?
4. Do you know if your digestion protocols impaired a little your surface proteins?
That is a very good question. We used Liberase™ and it is possible that it dissociated surface proteins. This is an area for further study.
5. Aren't you afraid that the cryopreservation may be more deleterious for some cell subtypes than others?
Yes, I am. Not just cryopreservation, but also tissue processing for single cell seq. That is why I do not think CITE-seq can be used to accurately quantify the number or relative proportions of cells in a tissue. All we can say is that there are at least 8 cell populations, 5 of which express high levels of COL1A1/2. There may well be others.
6. How near is this technique to be used for diagnosis?
It is currently an expensive test and that, combined with a lack of clarity in how we define the various stages of tendinopathy (these were all deliberately end stage) and no treatments, probably means it still has more value as a research tool.
7. How did you decide the number of hashing-tags? Did you need to set up a pilot study for 8?
I wanted the most possible and at the time that was 8. This number is well within the technical constraints and since our overall cell yield is low, I could even use more than 8 hashed samples in a single lane. Similarly, the only limit on surface TotalSeq™ mAb is physical "binding space" as opposed to flow cytometry, where it is the visual spectrum.