Cell Prep Recommendations
Guidance on preparing different cell types for the assay
General Guidelines
PBMCs
Primary tissue and organoids
General guidelines
Before the Cell Priming step
- Ensure a viability of >= 90% before starting our assay
- Count cells accurately (>10% difference between counts) to ensure correct cell input
- Filter cells to minimize clumping
|
Cell category |
Washed CPair volume |
Cell input |
CPM concentration |
|
Cell line |
15 µL (+ 5 µL PBS) |
5,000 |
1X |
|
PBMC |
20 µL |
8,500 |
1X |
|
Organoid |
20 µL |
10,000 |
1.5X |
|
Primary tissue |
20 µL |
10,000 |
2X |
PBMCs
- Our assay in not compatible with cell preparation that carries through any paramagnetic beads to the incubation with CPM. For example, our assay is not compatible with positive immunomagnetic sorting of PBMCs. Only use negative sorting where the unlabelled supernatant contains the desired cells
Primary tissue and organoids
Primary tissue and organoids often release a lot of debris after dissociation. Protein and lipid residues severely impact the efficiency of our assay and care must be taken to minimize them. Here are some suggestions to reduce debris in solution:
- Perform a debris removal step if there are sufficient cells
- Use large volume washes after dissociation (± 5 mL)
- Perform 2-3 washes with PBS + 0.04% BSA before counting and cell priming
- Perform 2 filtering steps, ideally a 70 µm filtering step before the washes, and a filtering step appropriate for your cell size (eg. 40 µm) before counting
- Perform an AO/PI stain and check cells under the microscope to ensure debris removal
To reduce cell loss during extra washes
- Use a centrifuge with swinging buckets instead of fixed angles during cell washes to pellet cells better
- Increase centrifugation time where possible