Introduction
ioMicroglia are human induced pluripotent stem cell (iPSC)-derived microglia, precision reprogrammed using opti-ox™ technology. Like all primary human microglia cells, ioMicroglia are adherent cells that form attachments to their surrounding environment. For many applications—including flow cytometry—it is necessary to break these attachments and bring the microglia into suspension. However, doing so is a delicate process that, if done too forcefully, may damage the cells or cause them to clump together. To ensure microglia remain viable, it is essential to perform gentle detachment from the culture dish. This process often involves loosening the cells with a solution containing proteases, chelating agents, or collagenases, such as Trypsin/EDTA® or Accutase®, followed by gentle washing or manual scraping of the culture dish surface.
Prolonged incubation with the detachment solution can be detrimental for cells, therefore it’s beneficial to neutralise the solution after detachment using serums that are common in culture media. Once in suspension and washed of detachment solution, cells are ready for replating, cell sorting, or staining for flow cytometry (among many other applications).
Protocol overview
Materials and equipment
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ioMicroglia cells (cat no: ioA021) cultured for at least 24 hours post-thaw
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Accutase
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ioMicroglia basal medium – as described in the ioMicroglia user manual
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15mL tubes
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Phosphate Buffered Saline (PBS)
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Biological safety cabinet
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Standard tissue culture wares (pipettes, tips, culture plates)
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Normoxic cell culture incubator (37°C, 5% CO2)
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Bench top centrifuge
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Brightfield microscope
Please request the ioMicroglia user manual from technical@bit.bio if required.
Protocol
1. After the ioMicroglia have been cultured for at least 24 hours post-thaw, gently aspirate all medium from wells, being careful not to dislodge any ioMicroglia from the surface of the plate.
2. Gently add Accutase to the culture vessel. We recommend using 10mL for each 95cm2 of the surface area outlined in the table below:
Table 1: Accutase volumes
|
Plate format |
Surface (cm2) |
mL/well |
Volume/well of basal medium to dilute |
|
6 well |
9.5 |
1 |
100µL |
|
12 well |
3.8 |
0.5 |
50µL |
|
24 well |
1.9 |
0.25 |
25µL |
3. Incubate the plate at 37°C for 4 minutes.
4. Using a brightfield microscope, check whether the cells are starting to dissociate from the surface of the plate. The microglia will start to lift off the plate when they are dissociating. If they are not detaching, return to the incubator for a further 2 minutes.
5. Once detached, use a micropipette to gently resuspend the cells in the Accutase solution. Transfer the resuspended cells to an appropriately labelled 15mL tube.
6. Dilute the Accutase and cell suspension with 10% ioMicroglia basal medium and mix gently, to neutralise the Accutase.
7. Centrifuge tubes at 330 xg for 4 minutes.
8. Remove the supernatant and resuspend the pellet in 200µL PBS.
9. The suspended cells should be promptly used for flow cytometry or other single cell experiments.
Application support
If you have any questions or need assistance, please reach out to technical@bit.bio and we will do our best to support you.
Published August 2023, version 3