- [Resource Hub](/) - [Field Guide](/?resource-type=field-guide#library) - TIL Counting and Immune Cell Killing: Dual-Cassette Optimization Guide # TIL Counting and Immune Cell Killing: Dual-Cassette Optimization Guide The Bottom Line Up Front: TIL counting and immune cell killing assays are immediate applications for dual-cassette workflows. Cancer cells are large, T cells are small - no single cassette optimizes both. Run S+ or S for accurate T cell counts, M+ or M for accurate tumor/target counts. The extra run takes minutes but delivers publication-quality E:T ratios and killing percentages. WHICH CASSETTES FOR YOUR INSTRUMENT Moxi V and Moxi GO II use S+ and M+ cassettes. Moxi Z uses S and M cassettes. Same sizing principles, same selection logic — just match the cassette type to your instrument. All recommendations in this guide apply across the Moxi family. ## Why Immuno-Oncology Demands Dual-Cassette Workflows Immuno-oncology applications inherently combine cells from opposite ends of the size spectrum. This isn't a corner case - it's the fundamental nature of the field. T cells are 6-10 micrometers, NK cells are 10-12 micrometers, and tumor cells are 15-30+ micrometers. Single-cassette measurement forces compromise: either undercount small lymphocytes (M+ cassette with weak signals) or risk clogging on tumor cells (S+ cassette with oversized cells). Neither gives you accurate data for both populations. ### TL;DR - Immuno-Oncology Workflow Essentials - TIL counting: T cells (6-10 μm) + tumor cells (15-30 μm) = mandatory dual-cassette application - Immune killing assays: small effectors + large targets = same dual-cassette requirement - S+ or S for lymphocytes/effectors, M+ or M for tumor/target cells - Calculate E:T ratios from optimized individual measurements, not compromised single-cassette data - Single-cassette shortcuts introduce systematic error into every ratio calculation ## Immuno-Oncology Dual-Cassette Protocols Complete workflows for TIL counting, cytotoxicity assays, and accurate ratio calculations. Why Immuno-Oncology Demands Dual-Cassette Workflows Immuno-oncology applications inherently combine cells from opposite ends of the size spectrum. This isn't a corner case - it's the fundamental nature of the field: T cells: 6-10 micrometers - firmly in S+ cassette territory NK cells: 10-12 micrometers - still within S+ optimal range Tumor cells: 15-30+ micrometers - requires M+ cassettes TIL counting represents one of the immediate applications for dual-cassette workflows. The same is true for all immune cell killing assays: cancer cell big, your T cell small. Single-cassette measurement forces compromise: either undercount small lymphocytes (M+ cassette with weak signals) or risk clogging on tumor cells (S+ cassette with oversized cells). Neither gives you accurate data for both populations. TIL Counting: Complete Workflow Tumor-infiltrating lymphocyte quantification requires accurate counts of both infiltrating T cells and tumor cells to calculate meaningful infiltration ratios: 1 Prepare single-cell suspension from tumor sample (enzymatic or mechanical dissociation) 2 Run first aliquot with S+ or S cassette - record T cell count and size distribution 3 Run second aliquot with M+ or M cassette - record tumor cell count and size distribution 4 Calculate infiltration ratio: TIL count (S+) / tumor count (M+) 5 Report optimized values for each population with cassette method documented The extra cassette change and measurement takes a few minutes. The alternative - systematic error in one or both populations - corrupts all downstream analysis. Immune Cell Killing Assays: Protocol Optimization Cytotoxicity assays quantify immune cell killing through effector:target ratios and surviving target counts. Accurate counts of both populations are essential: #### CAR-T Cytotoxicity CAR-T cells (8-12 μm) vs tumor target cells (15-25 μm). Set up E:T ratios using S+ counts for effectors, M+ counts for targets. Track killing by M+ target cell counts over time. #### NK Cell Killing NK cells (10-12 μm) vs cancer cell lines (15-30 μm). Dual-cassette workflow captures both populations optimally for accurate killing percentage calculations. #### ADCC Assays Effector cells (PBMCs, 8-15 μm) plus antibody vs target cells (tumor lines, 15-25 μm). S+ for effector quantification, M+ for target survival assessment. All immune cell killing assays share this structure: small effector, large target. Dual-cassette workflows aren't optional for publication-quality data. Calculating Ratios from Dual-Cassette Data Proper calculation uses the optimized count from each cassette measurement: E:T Ratio Calculation: E:T Ratio = Effector count (S+ cassette) / Target count (M+ cassette) Killing Percentage: % Killing = [1 - (Targets at T x / Targets at T 0 )] × 100 Both target counts from M+ cassette measurements at different timepoints. Why This Matters Single-cassette measurements introduce bias in one population. If you undercount effectors (M+ cassette for small cells), your E:T ratio is artificially low. If you undercount targets (S+ clogging on large cells), your killing percentage is artificially high. Both errors propagate through all calculations. Documentation and Method Reporting For publications and regulatory submissions, document your dual-cassette methodology clearly: Methods section language: "Cell counts were obtained using Moxi [model] with cassette selection optimized for cell size. Lymphocyte/effector populations were quantified using S+ cassettes (optimal for cells 15 μm). E:T ratios were calculated using optimized counts from each population." Supporting rationale: Reference the Coulter principle physics and cell-to-aperture optimization. This isn't an unusual method - it's the technically correct approach for heterogeneous populations. Data presentation: Include cassette type in your data tables. Reviewers should see that you measured each population appropriately, not that you forced compromise on one or both. ## Troubleshooting Guide T cell counts seem low in tumor digests Solution: Verify you're using S+ or S cassettes for T cell quantification. Small lymphocytes generate weak signals in M+ or M cassettes, causing undercounting. Clogging when counting tumor samples Solution: Use M+ or M cassettes for tumor cell counts. Large tumor cells clog S+ or S apertures. If clogging persists, verify dissociation quality - clumps clog even appropriate apertures. E:T ratios don't match expected setup Solution: Check that you're using S+ for effector counts and M+ for target counts. Using the same cassette for both introduces systematic bias that skews ratios. Killing percentages seem artificially high Solution: If target cells are clogging S+ cassettes, you're undercounting survivors. Switch to M+ for target counts to get accurate surviving cell numbers. ## Frequently Asked Questions How do I accurately count TILs in tumor samples? TIL counting requires dual-cassette workflows because T cells (6-10 micrometers) and tumor cells (15-30+ micrometers) span both sides of the 15-micrometer boundary. Run with S+ cassettes for accurate T cell quantification, then M+ cassettes for tumor cell counts. This gives you optimized counts for both populations to calculate accurate infiltration ratios. What cassettes should I use for immune cell killing assays? Immune cell killing assays pair small effector cells (T cells, NK cells at 6-12 micrometers) with large target cells (cancer cells at 15-30 micrometers). Use S+ cassettes for effector cell counts and M+ cassettes for target cell counts. This dual-cassette approach provides accurate E:T ratios and killing percentage calculations. How do I calculate E:T ratios from dual-cassette measurements? Use the optimized count from each cassette: effector counts from S+ measurements, target counts from M+ measurements. Calculate E:T ratio = (effector count from S+) / (target count from M+). This gives more accurate ratios than any single-cassette compromise measurement could provide. Can I use a single cassette for immune cell killing assays? Technically yes, but with significant accuracy trade-offs. M+ cassettes will undercount small effector cells, inflating apparent E:T ratios. S+ cassettes risk clogging with large target cells and may undercount them. Dual-cassette workflows take extra minutes but provide accurate counts for publication-quality data. ### Key Takeaway Immuno-oncology applications are built on size mismatches between effector and target cells. TIL counting, CAR-T cytotoxicity, NK killing, ADCC - all require accurate counts of both small and large cells. Dual-cassette workflows using S+ or S for lymphocytes and M+ or M for tumor cells deliver the accurate ratios your research depends on. The alternative isn't "good enough" data - it's systematically biased data that propagates error through every calculation. [Back to all resources](/#library) ## Similar resources [App Note](https://precisioncellsystems.com/wp-content/uploads/2025/08/Moxi-Average-Cell-Size-By-Cell-Type.pdf) Moxi GO II Moxi V Moxi Z 2025 ### Moxi - Average Cell Size By Cell Type [Download App Note](https://precisioncellsystems.com/wp-content/uploads/2025/08/Moxi-Average-Cell-Size-By-Cell-Type.pdf) [Field Guide](/resources/05-suboptimal-resolution/) Moxi GO II Moxi V Moxi Z 2026 ### The Suboptimal Resolution Problem: Why Your Cell Populations Look Merged Using an aperture much larger than necessary reduces sizing resolution by creating smaller signal differences between cell sizes. Cell populations that should be distinguishable appear merged when the aperture is too large for the cells being measured. Target 15-40% of aperture diameter for optimal resolution. If you're counting lymphocytes on M+ cassettes because it works, you're sacrificing the sizing resolution that S+ cassettes would provide. [Read Field Guide](/resources/05-suboptimal-resolution/) [Field Guide](/resources/06-fifteen-micron-boundary/) Moxi GO II Moxi V Moxi Z 2026 ### The 15-Micrometer Decision: A Practical Cassette Selection Framework The 15 μm boundary provides clear selection criterion: cells under 15 micrometers use S+ cassettes, cells over 15 micrometers use M+ cassettes. This boundary isn't arbitrary - it's where each aperture size achieves the optimal 15-40% cell-to-aperture ratio for signal quality and sizing resolution. Know your cell size, follow the boundary, and cassette selection becomes automatic. [Read Field Guide](/resources/06-fifteen-micron-boundary/) [Field Guide](/resources/03-mixed-population-dilemma/) Moxi GO II Moxi V Moxi Z 2026 ### The Mixed Population Dilemma: When One Cassette Can't Capture Everything When your sample contains both small and large cells, no single cassette optimizes measurement for both populations. The solution: run the same sample twice - once with S+ to get accurate small cell counts, once with M+ to get accurate large cell counts. This dual-cassette workflow delivers accurate data for both populations rather than compromised data for everyone. [Read Field Guide](/resources/03-mixed-population-dilemma/) [Field Guide](/resources/04-coincidence-artifact/) Moxi GO II Moxi V Moxi Z 2026 ### The Coincidence Artifact: When Two Cells Count as One Coincidence - multiple cells in the aperture simultaneously - causes two cells to be counted as one, corrupting both count and size data. Optimal aperture utilization means targeting 15-40% of aperture diameter so cells generate strong signals while avoiding coincidence artifacts. Match your cassette to your cell size, stay within concentration guidelines, and coincidence becomes a non-issue. [Read Field Guide](/resources/04-coincidence-artifact/) [App Note](https://precisioncellsystems.com/wp-content/uploads/2026/02/Moxi-Applications-Compendium.pdf) Moxi GO II Moxi V Moxi Z 2026 ### Applications Compendium Scientists are concerned with speed, accuracy, and convenience, and those running the lab and industries are concerned with the cost typically associated with high-performing instruments. Our proprietary Coulter Principle-based system delivers on all three accounts, and is therefore a perfect fit for any cell biology benchtop. [Download App Note](https://precisioncellsystems.com/wp-content/uploads/2026/02/Moxi-Applications-Compendium.pdf) [Tech Note](https://precisioncellsystems.com/wp-content/uploads/2025/09/ORFLO-Single-or-Dual-Color-Viability-Staining-Tech-Tip.pdf) Moxi GO II Moxi V 2025 ### Moxi Single or Dual Color Viability Staining Tech Note [Read Tech Note](https://precisioncellsystems.com/wp-content/uploads/2025/09/ORFLO-Single-or-Dual-Color-Viability-Staining-Tech-Tip.pdf) [Tech Note](https://precisioncellsystems.com/wp-content/uploads/2025/09/ORFLO-Nuclei-Counting-Tech-Tip.pdf) Moxi GO II Moxi V 2025 ### Moxi Nuclei Counting Tech Note [Read Tech Note](https://precisioncellsystems.com/wp-content/uploads/2025/09/ORFLO-Nuclei-Counting-Tech-Tip.pdf) [Tech Note](https://precisioncellsystems.com/wp-content/uploads/2025/09/ORFLO-PBMC-Counting-and-Viability-Tech-Tip.pdf) Moxi GO II Moxi V Moxi Z 2025 ### Moxi PBMC Counting and Viability Tech Note [Read Tech Note](https://precisioncellsystems.com/wp-content/uploads/2025/09/ORFLO-PBMC-Counting-and-Viability-Tech-Tip.pdf) [Field Guide](/resources/01-optimization-burden/) Moxi GO II Moxi V 2026 ### The Optimization Burden: Hours Wasted on Viability Protocol Development Every hour spent optimizing viability dye concentrations is an hour not spent on your actual experiments. It's optimized for use - that's the important thing. You don't have to optimize it as a customer. Pre-optimized viability reagents eliminate the titration experiments, the incubation testing, the cell-type-specific protocol development. Why spend time optimizing when validated performance is available from the first use? [Read Field Guide](/resources/01-optimization-burden/) [Field Guide](/resources/02-unknown-product/) Moxi GO II Moxi V 2026 ### The Unknown Product: Most Moxi Users Don't Know This Exists Most of the issue with viability reagents is that most people don't even know they exist. If you're running viability assays on Moxi V or Moxi GO II with generic dyes you optimized yourself, there's a better option you may not have heard about: pre-optimized, ready-to-use viability reagents designed specifically for your instrument. Now you know. [Read Field Guide](/resources/02-unknown-product/) [Field Guide](/resources/04-protocol-hunt/) Moxi GO II Moxi V 2026 ### The Protocol Hunt: Searching for Methods That Already Exist Searching for viability protocols, adapting literature methods, trial-and-error until something works - this is time you don't need to spend. The user manual is designed to be super easy. Concentration-based instructions tell you exactly what to do: put X amount of viability reagent and put X amount of sample, incubate and go. No protocol hunting required. [Read Field Guide](/resources/04-protocol-hunt/)