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We first determined optimal channels (per marker) based on antigen abundance. Next, we determined antibody concentration by testing five different antibody concentrations in a serial dilution. Depending on the nature of the marker, healthy cells were either unstimulated or stimulated with an appropriate biological stimulus, such as PHA or PMA. Finally, spillover was assessed by looking at signals in other channels. To confirm the panel works to detect all major immune cell populations, frequencies of all major immune cell lineages and subsets were measured in PBMCs from 8 healthy subjects.

This is described in finer detail in the Antibody validation section beginning on page 16 of our Panel Validation Whitepaper.

Using a process very similar to how we validated our base panels, we verify the optimal staining concentration at which each metal-conjugated antibody demonstrates a detectable and accurate signal on the mass cytometer, while minimizing background signal and spillover into other channels. Once titrated and validated individually, they are added to the panel and the full panel is then validated.

We’ve validated 158 unique antibodies corresponding to 111 human, 62 mouse, and 47 non-human primate markers due to cross-reactivity of some antibodies. The full list can be found in our “In-house Validated Antibodies” downloadable spreadsheet.

Yes, we can work with any monoclonal antibody in a protein-free format (i.e. without BSA or other added proteins). Metal conjugation is performed in-house and the newly metal-conjugated antibody is validated on in-house PBMC samples as described previously.

If your marker of interest is not expressed on PBMCs, a cell line or other sample type that expresses the marker should be provided or can be purchased for panel validation purposes.

Typically 2 weeks from receipt of all antibody reagents. For some cases, such as those where stimulation is required for marker expression, antibody validation can take up to 4 weeks and this will be communicated in advance.

All antibodies are validated in-house by our team, not outsourced. For new antibodies, pre-conjugated antibodies are used when commercially available. If conjugation is required, it is performed in-house by our team, not outsourced, to ensure high performance and fast turnaround. All new antibodies, whether pre-conjugated or conjugated in-house, are validated by our team.

Currently, we do not support the addition of peptide tetramers into our panels.

Yes, conditionally, and no.

Yes – Transcription factors and other highly expressed intracellular markers can easily be detected. Our base panels typically include transcription factors such as Foxp3, T-bet, and TCF1 and intracellular functional proteins like Granzyme B and KI-67. We have also validated a number of other transcription factors like HIF1a and RORgt, intracellular proteins like Perforin and X, and even metabolic markers like mTOR and GAPDH.

Conditionally – Some cytokines, such as IFNg and TNFa, can be detected in activated cells without additional stimulation. However, most cytokines typically require cell stimulation in the presence of a secretion blocker (like Brefeldin A) in order to build up sufficient protein for detection. Our scientists can help you determine whether prior stimulation may be necessary depending on your cytokine of interest.

No – Chemokines are often produced in low quantities not well-suited for cytometry based detection. While chemokine production cannot be easily detected, chemokine receptor expression is detectable and can provide information about which immune subsets are responsive to chemokine gradients.

At Teiko Bio, we’ve worked with a wide range of sample types across human, mouse, and non-human primates. This includes PBMCs, whole blood, bone marrow aspirate, TILs, cell therapy product, and even dissociated fresh tumor and tissue. For a full list of sample types, download our Sample Guidelines.

For prospective studies, we recommend collecting whole blood into standard vacutainers containing, in order of preference, heparin, citrate, or EDTA and using our simple two-step, 20 minute TokuKit for on-site processing. This process is faster, easier, and cheaper than PBMC isolation, has no centrifugation steps, and yields high-quality cytometry data.

For retrospective studies, we accept viably cryopreserved PBMCs or whole blood samples that have undergone red blood cell lysis and fixation using PROT1, or Stable-Lyse, Stable-Store.

We cannot accept whole blood collected using an RNA or DNA isolation tube, nor serum samples as these lack intact cells required for cytometry-based analyses.

Our internal R&D data shows that with Whole Blood you get the same quality information about non-granulocytes as with PBMCs, plus additional insights into granulocyte populations (neutrophils, eosinophils, basophils). Teiko scientist Dr. Jolien Sweere reviews the data in our webinar: Whole blood or PBMCs for immune profiling?

Our validation data show the minimum input for accurate detection of all major immune cell populations is 50K viable cells. This equates to a minimum of about 100 µL of whole blood. Minimum tumor and tissue sample size depends on immune infiltration, however a sample volume of 350 mm3 should yield a sufficient number of immune cells for analysis [Allen et al., 2020]. While these are the minimum volumes for detection, we recommend 2mL of whole blood or 1 million viable PBMCs for most samples.

No, sample collection on CyTOF is a destructive process, meaning the cells cannot be recovered after acquisition.

Yes, please let us know when planning your project how much of each sample you would like us to return and where to ship the remaining samples. Thawed PBMC samples can be separated into aliquots and viably cryopreserved for return shipment.

Yes, if samples arrive at our facility within 48 hours of collection. Blood stored at room temperature for 24 to 48 hours shows modest changes in marker expression, but dramatic changes by 72 hours. Temperature fluctuations during shipping and delivery delays are other factors that can impact fresh blood samples. For this reason, we highly recommend fixing blood on-site using our TokuKit and shipping samples on dry ice to preserve marker expression and sample quality during shipping.

At the moment we only include short-term storage of samples (less than 6 months) as part of an analysis project with Teiko. Long-term storage of samples (greater than 6 months) can be added for a nominal fee to a larger analysis plan for ongoing and prospective clinical trials. Cryopreserved samples are stored in liquid nitrogen and fixed samples are stored at -80°C.