Cell Culture Hub
Analyze the before-and-after AVITI24™ run images to identify optimal cell confluency for better experimental outcomes.
Overview
The Cell Culture Hub guides Teton™ users in optimizing cell culture parameters, such as seeding density and surface coatings, in a plate-based format before preparing samples on a Teton Slide Kit. Teton users can compare their bright field and AVITI24 images with Cell Culture Hub images from a similar preparation to assess cell confluency and adjust culture parameters for optimal results. The About the Experiment section explains how these images were generated. This optimization protocol is recommended for users who are new to Teton or are working with unvalidated cell types. For optimization protocol details, refer to the Teton Optimization and Screening User Guide and Teton Cell Culture Optimization Tech Note. For instructions on sample preparation for a Teton CytoProfiling run, consult the Teton CytoProfiling User Guide. The Knowledge Base includes helpful videos and additional tips on cell handling.
About the Experiment: Suspension - Jurkat
Surface Coating:
Prior to cell culture steps, CellVis 96-well plates (Cellvis catalog # P96-1.5P), Glass-bottom 96-well plates (Ibidi catalog # 89627), and 12-well Teton Slide Kits (Part # 860-00032) were custom coated with Poly-L-Lysine (PLL) as detailed in the Teton CytoProfiling User Guide (MA-00053).
Cell Culturing:
- Jurkat cells were cultured with complete RPMI-1640 in a stationary T225 flask to ensure sufficient cell count for the optimization experiment.
- Cells were gently pipette-mixed to break any clumps that may form in the culture during growth.
- Cells were counted using a hemocytometer to determine the approximate cell concentration in the culture.
- An aliquot containing the appropriate number of cells was taken from the culture flask. The cells were centrifuged at 200 x g for 5 minutes.
- The cell pellet was resuspended in 1x PBS to an appropriate concentration for the highest cell concentration. The cell suspension can be recounted to confirm starting concentration for optimization titration.
Cell Seeding Optimization:
- For the cell concentration titration, each concentration was diluted individually with 1x PBS in an intermediate tube before seeding into the plate and slide kit wells. This ensures a uniform cell suspension and even distribution of the culture during seeding.
- Each concentration was seeded in replicates across both the plate and slide kit. The seeding volumes are listed below for each configuration used:
| Vendor | Layout | Well Shape | Well Area | Seeding Volume per Well |
|---|---|---|---|---|
| CellVis | 96-well Plate | Circle | 30 mm² | 100 µL |
| Ibidi | 96-well Plate | Square | 7.4 mm x 7.4 mm | 150 µL |
| Element Biosciences | 12-well Slide Kit | Squircle | 7 mm x 7 mm | 150 µL |
The seeding concentrations, listed as cells per well in 150µL seeding volume, for both the for 96-well plate and 12-well Slide Kit are listed below:
| Row | 96-well plate | 12-well Slide Kit |
|---|---|---|
| A | 500,000 | 300,000 |
| B | 300,000 | 300,000 |
| C | 240,000 | 120,000 |
| D | 180,000 | 120,000 |
| E | 120,000 | 30,000 |
| F | 60,000 | 30,000 |
| G | 30,000 | -- |
| H | 10,000 | -- |
Fixation and Downstream Applications:
- The plate and slide kit were imaged using bright field microscopy at a 40X magnification before fixing the cells.
- Cells were fixed to the surface using 8% formaldehyde as described in the Teton Optimization and Screening User Guide to attach and fix suspension cells and imaged using bright field microscopy again after fixation.
- Assessment of sample preparation was performed using the Onboard Cell Paint Imaging Kit (Part # 860-00047) or a full Teton Immuno Panel Kit (Part # 830-00039).
- Bright field microscopy and AVITI24 results were used to determine the optimal seeding density for the Teton workflow.
If you have any questions on how to seed your cells for your experiment, contact Element Support.