CyTOForum

Hi Jahangir,

For barcoding, I would recommend using a minimum number of channels required to achieve the barcoding level that you need. This is because in my testing (and in most antibody-based barcoding schemes, including the CD298/b2m/CD45 pioneered by Felix), I only used one clone for the CD45 epitope. Therefore, each CD45 barcode reagent will be in competition with each other CD45 barcode reagent. Henrik showed this effect nicely in his first barcoding paper, where going from 1 to 2 to 3 barcode channels per barcode had a stepwise decrease in barcode channel signal intensity (and therefore resolution of the barcode). Even in my own data presented earlier, you can see that 1/6 barcodes are almost 1e3, while 3/6 barcodes are lower at just above 1e2. Additionally, the impurity spillovers are additive the more barcodes you use, thus increasing your background.

For example, 6 BC channels would give you 1/6/15/20/15/6/1 possibilities for 0/1/2/3/4/5/6-of-6 codes (remember, Pascal's triangle is a quick way to calculate this). So, if you need 15 BCs, you can do 2-of-6 or 4-of-6. 2-of-6 would save you on reagents, but also have higher signal in each of the channels you used than would the corresponding 4-of-6. Of course, if you wanted 20 barcodes, you have to do 3-of-6 (assuming 6 BC channels).

As such: 7 BC channels give you 1/7/21/35/35/21/7/1 for 0/1/2/3/4/5/6/7-of-7 codes. If you do 3-of-7 or 4-of-7, you get the same number of BC plexing.

Therefore, I don't see why you would do 4-of-7: you a) don't get any increase in barcoding plexing, b) have more reagent cost, and c) have lower signal (lower resolution due to decrease in Pos and potential increase in Neg) per BC channel. To me, these issues don't sufficiently offset any theoretically minor increase in "purity" (especially the lower resolution issue).


Mike

We have also started conjugating with the Cadmiums. We have done 3 separate batches of conjugations and across sessions we got unimpressive yields ranging between 15-30%, while with X8 conjugations we get 40-70% yield. The Fluidigm user guide says expect 10% lower than for lanthanides but we are getting 20-40% lower. How are people getting on for yields?
That aside, the labelling has worked. We are sticking to high abundance surface markers such as CD8 and beta2m. Cd116 conjugates have impressive high intensity staining (>300 ion counts) while Cd106 much lower (<100 ion counts).
NK

Hi Naeem,

I've now done 12 conjugations with the Cd MCP9 kits (basically, I've conjugated on two separate occasions each of the 6 kits I purchased).

The first six were all about 70% recovery.

The second six, 4 were 70%, one was 60%, the other was 55%. No idea why: same CD45 clone, and I had even pooled and then resplit the MCP9 before adding the separate Cd isotopes.


Mike

We've done a total of 8 cadmium conjugations and our recoveries have also been somewhat variable. Perhaps a little lower, but not too far off from what we typically see when conjugating 100ug of antibody with the X8 kits:

B2m, clone 2M2
111Cd: ~43%
112Cd: ~47%
114Cd: ~49%
116Cd: ~58%

CD298, clone REA217
111Cd: ~71%
112Cd: ~62%
114Cd: ~70%
116Cd: ~58%

We've been finding that these work really well for live cell barcoding (higher signal/better barcode separation than we were typically seeing when conjugating the same clones to Pt using monoisotopic cisplatin).

I'd also caution folks to actually read the Fluidigm protocol for the MCP9 kits since there are some minor differences from the X8 kits that we hadn't initially noticed when we first skimmed through it (e.g., use of a 100kDa instead of 50kDa filter for the final washes, and recommendation of a different antibody stabilization buffer).

Has anyone modified the Cadmium protocol or are you all following the Fluidigm protocol? We had a recent round of conjugations that had a very low recovery less than 30% and the mabs had poor to no signal. Two of the mabs are CD4 and CD8, know what they should look like. One of our technicians, no longer with us, had labeled these mabs/clones before with the Cadmium kits successfully. Recovery within range and strong signal. Not sure if he altered protocol or followed all of Fluidigms steps.

When we get poor recovery and the antibody does not seem to bind/work is there a particular reagent that could be the issue? Any troubleshooting suggestions greatly appreciated.

Thanks
Mike

Hi Mike,

I follow the Fluidigm protocol for Cadmium/MCP9 without any real modifications (I think I might cut out a wash late in the protocol, but definitely no changes during the loading and conjugation).

I've had a random conjugation fail (like occasionally happens with X8/DN3), but not a whole group of them.


Sorry I can't be much help,
Mike