Flow Cytometry Services

Flow cytometry measures the properties of individual cells rather than the average across a population, which is what makes it indispensable in immunology. A tumor lysate reporting elevated CD8 protein cannot distinguish a modest increase across many cells from a large increase in a few. Flow cytometry resolves that directly, quantifying how many cells of each type are present and what state each is in.

Altogen Labs provides flow cytometric analysis of blood, tumor, spleen, bone marrow, and other tissues from in vivo studies, together with analysis of cultured cells. It is the primary method for immune profiling in immuno-oncology programs and for verifying reconstitution in humanized mouse models. To discuss a panel, request a quote.

Panel design

Panel design is where a flow study succeeds or fails. Markers are selected to answer the specific question rather than to characterize everything available, since each additional parameter adds spectral overlap that must be compensated and reduces the resolution available for the markers that matter. A focused panel answering one question well is more useful than a broad panel answering several poorly.

Fluorophore assignment follows marker abundance: dim markers are assigned bright fluorophores, and markers expected to co-express are assigned to channels with minimal spectral overlap. Compensation controls are prepared for every fluorophore in the panel, and fluorescence minus one controls are used where a gate boundary is not clearly bimodal, because drawing that boundary by eye on a continuous distribution is the most common source of irreproducible flow data.

Immune profiling

Standard immune profiling covers human CD45 for reconstitution assessment, CD3, CD4, and CD8 for T-cell populations, CD19 for B cells, CD56 for natural killer cells, CD14 for monocytes, and activation and checkpoint markers as the mechanism requires. Panels are extended to memory and differentiation subsets, exhaustion markers, and regulatory populations where these are central to the question.

In humanized studies, reconstitution is verified against defined eligibility thresholds before animals are randomized to treatment, so that immune status is balanced across arms rather than assessed retrospectively as a covariate.

Tissue handling constraints

Flow cytometry imposes handling requirements that must be settled before a study opens, because they cannot be satisfied afterward. Most panels require fresh or promptly processed material, and tissue frozen for molecular endpoints is generally unusable for surface phenotyping. Tumor dissociation method affects which populations survive and which epitopes remain detectable, and an aggressive protocol can deplete precisely the population of interest.

Where a study requires both flow cytometry and frozen tissue from the same tumor, the division is planned at necropsy with the flow aliquot processed first.

Applied examples

Reconstitution verification. Peripheral blood from humanized animals is assessed for human CD45, CD3, and CD4 to CD8 ratio against eligibility criteria, with animals below threshold excluded before randomization rather than contributing variance to the study.

Intratumoral immune response. Tumors from treated and control animals are dissociated and profiled for effector and regulatory populations, establishing whether an antitumor effect is accompanied by the immune changes the mechanism predicts.

Cell therapy persistence. Circulating engineered cells are quantified at intervals following administration, distinguishing durable engraftment from transient expansion followed by loss.

Acquisition standards and data reporting

Instrument setup, compensation matrix, gating strategy, and the number of events acquired are defined before samples are run and held constant across the study, since a gating strategy adjusted between timepoints makes the resulting frequencies incomparable. Gating is performed to a documented strategy applied uniformly rather than adjusted per sample. Reports include the gating strategy, representative plots, per-animal population frequencies and absolute counts where relevant, and the raw data files.

Send the populations of interest, tissue type, and study design, or request a quote.