CyTOF comp beads-best practices questions
Hi all,
We haven't yet adopted capture bead compensation (like in the initial Chevrier paper, or updated in the Os-labeled Budzinski paper). But I was recently talking with someone analyzing a dataset with comp data, and a couple questions came up.
1. In this dataset, there were 2 panels, so 2 sets of Comp bead files. These comp bead samples were run separately from the cells (no Os-labeling to distinguish). One file appeared to contain EQ beads (Ce140 signal, but no Ce140-labeled antibody), while the other did not.
- This seemed odd to me: shouldn't you include EQ beads to first norm your Comp before doing the Comp (just in case your machine was behaving differently)?
- looking back at the Chevrier paper ( https://doi.org/10.1016/j.cels.2018.02.010 ), there is no statement of including EQ norm beads: "Briefly, for each channel assessed in the panel, one full drop of BD Compbead was loaded in a well of a v-bottom 96 well plate and stained with 1 μg of the corresponding metal-labeled antibody. Beads were stained for 15 min at room temperature. After staining, beads were washed three times in CSM and then pooled in a single tube. Beads were then fixed in 1.6% PFA/PBS for 1 h at room temperature. After fixation, beads were washed twice in PBS and twice in water. Before acquisition, beads were resuspended in 500 μL of water. Bead and cell data were acquired on a Helios mass cytometer (Fluidigm) using instrument-based dual-count calibration, noise reduction, and randomization."
- in the Budzinski paper, because of the Os-labeling, the comp beads could be pooled with the cells and run together, so EQ beads were also present because of the cells: "For the experiment shown in Fig. 3, a large batch of beads was labeled with OsO4, aliquoted into cavities of a 96-well Deep Well Plate (Corning, Corning, NY), and incubated with individual Ab conjugates (Supplemental Table I). After loading of beads with Ab conjugates and washing, bead aliquots were pooled and either stored at −80°C (Supplemental Fig. 1B) or directly combined with cell suspensions previously stained with Intercalator-Ir and the same set of isotope-tagged Abs (1:4, v/v) for simultaneous acquisition."
2. Also in this dataset, 7 batches were run in a relatively short period of time (a few weeks), and then an 8th batch was run a few months later. As far as we can tell from Comp bead FCS file header timestamps, they only acquired comps once, during Batch 1 of the 7 batches.
- This also seems odd: it seems weird enough that they wouldn't acquire an aliquot of Comps with each day's acquisition, but it seems even weirder that months later they would reuse the same Comp file.
- So, that leads to my next question: for those people who are regularly performing CyTOF compensation, how often are you running a Comp bead sample (separate or spike-in)?
- I would have assumed that people are doing this *each day*, but I know even in the fluorescence flow world, people don't always do that.
- Note: "do you run comp beads with every experiment?" is different than "do you prep comp beads with every experiment?"; Budzinski et al state "Importantly, once prepared, compensation bead pools were storable at −80°C for at least 2 wk without affecting the signal intensity of the captured Ab conjugates (Supplemental Fig. 1B), simplifying its use as a routine spike-in compensation control in mass cytometry."
Mike
We haven't yet adopted capture bead compensation (like in the initial Chevrier paper, or updated in the Os-labeled Budzinski paper). But I was recently talking with someone analyzing a dataset with comp data, and a couple questions came up.
1. In this dataset, there were 2 panels, so 2 sets of Comp bead files. These comp bead samples were run separately from the cells (no Os-labeling to distinguish). One file appeared to contain EQ beads (Ce140 signal, but no Ce140-labeled antibody), while the other did not.
- This seemed odd to me: shouldn't you include EQ beads to first norm your Comp before doing the Comp (just in case your machine was behaving differently)?
- looking back at the Chevrier paper ( https://doi.org/10.1016/j.cels.2018.02.010 ), there is no statement of including EQ norm beads: "Briefly, for each channel assessed in the panel, one full drop of BD Compbead was loaded in a well of a v-bottom 96 well plate and stained with 1 μg of the corresponding metal-labeled antibody. Beads were stained for 15 min at room temperature. After staining, beads were washed three times in CSM and then pooled in a single tube. Beads were then fixed in 1.6% PFA/PBS for 1 h at room temperature. After fixation, beads were washed twice in PBS and twice in water. Before acquisition, beads were resuspended in 500 μL of water. Bead and cell data were acquired on a Helios mass cytometer (Fluidigm) using instrument-based dual-count calibration, noise reduction, and randomization."
- in the Budzinski paper, because of the Os-labeling, the comp beads could be pooled with the cells and run together, so EQ beads were also present because of the cells: "For the experiment shown in Fig. 3, a large batch of beads was labeled with OsO4, aliquoted into cavities of a 96-well Deep Well Plate (Corning, Corning, NY), and incubated with individual Ab conjugates (Supplemental Table I). After loading of beads with Ab conjugates and washing, bead aliquots were pooled and either stored at −80°C (Supplemental Fig. 1B) or directly combined with cell suspensions previously stained with Intercalator-Ir and the same set of isotope-tagged Abs (1:4, v/v) for simultaneous acquisition."
2. Also in this dataset, 7 batches were run in a relatively short period of time (a few weeks), and then an 8th batch was run a few months later. As far as we can tell from Comp bead FCS file header timestamps, they only acquired comps once, during Batch 1 of the 7 batches.
- This also seems odd: it seems weird enough that they wouldn't acquire an aliquot of Comps with each day's acquisition, but it seems even weirder that months later they would reuse the same Comp file.
- So, that leads to my next question: for those people who are regularly performing CyTOF compensation, how often are you running a Comp bead sample (separate or spike-in)?
- I would have assumed that people are doing this *each day*, but I know even in the fluorescence flow world, people don't always do that.
- Note: "do you run comp beads with every experiment?" is different than "do you prep comp beads with every experiment?"; Budzinski et al state "Importantly, once prepared, compensation bead pools were storable at −80°C for at least 2 wk without affecting the signal intensity of the captured Ab conjugates (Supplemental Fig. 1B), simplifying its use as a routine spike-in compensation control in mass cytometry."
Mike