Miltenyi Biotec solutions for pluripotent stem cell culture, passaging, and cryopreservation ensure gentle handling and recovery with high viability. In this application protocol, we introduce the use of these solutions, as well as a multicolor flow cytometry protocol that allows for the simultaneous quantification of intracellular and surface markers for easy phenotyping of human pluripotent stem cell cultures.
Maintain human embryonic stem cells (ES) or induced pluripotent stem cells (iPS) on a standard cell attachment matrix (e.g., Matrigel® or Laminin-521) in StemMACS™ iPS-Brew XF medium. The medium is designed to enable culture under feeder-free conditions and allows rapid culture initiation after cryopreservation. Follow the protocol of the medium data sheet.
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Use the StemMACS™ Passaging Solution XF for gentle detachment of pluripotent stem cell colonies and dissociation into cell clusters. The solution is designed to minimize manipulation of the culture, eliminating lengthy inactivation, dilution, or centrifugation steps. Thus, transfer into new cell culture conditions is reproducible, standardized, and fast, ensuring optimal viability and attachment. Follow the protocol of the solution data sheet.
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The panel for analysis of pluripotent stem cells includes antibodies for both surface markers and intracellular markers. Surface markers are stained first. Subsequently, cells are fixed and permeabilized for intracellular staining.
To set up the instrument and compensate for spectral overlap, single stainings for each antibody and an unstained cell sample are required. The unstained sample does not contain any antibody, but is otherwise treated in the same way as the stained samples regarding e.g. fixation and permeabilization. The pipetting scheme in the tables below provides an overview. As SSEA-1 is not expressed in pluripotent cells, the MACS® Comp Bead Kit, anti-mouse Igκ is used for compensation of PE-Vio® 770 instead of a cell sample.
▲ Note: Fluorescence-minus-one (FMO) controls might help you to set the gates more accurately. FMO controls contain all antibodies except for one.
Pipetting scheme for surface and intracellular staining. Cell surface staining refers to steps 3–8 of the protocol below. Intracellular staining refers to steps 14–19.
|Cell surface staining|
|Sample||PBE Buffer||Anti-TRA-1-60-PE||CD15-PE-Vio770, |
|1. Multicolor panel||85.6 µL||2.2 µL||2.2 µL||10 µL||10 µL|
|2. Unstained sample||100 µL||–||–||–||–|
|3. Single-color staining||107.8 µL||2.2 µL||–||–||–|
|4. Single-color staining||100 µL||–||–||10 µL||–|
|5.. Single-color staining||100 µL||–||–||–||10 µL|
|6. Single-color staining||100 µL||–||–||–||–|
|7. Single-color staining||100 µL||–||–||–||–|
|Sample||Inside Perm||Anti-Sox2-FITC||Anti-Oct3/4 Isoform A-APC|
|1. Multicolor panel||105.6 µL||2.2 µL||2.2 µL|
|2. Unstained sample||100 µL||–||–|
|3. Single-color staining||100 µL||–||–|
|4. Single-color staining||100 µL||–||–|
|5. Single-color staining||100 µL||–||–|
|6. Single-color staining||107.8 µL||2.2 µL||–|
|7. Single-color staining||107.8 µL||–||2.2 µL|
Isotype controls are used to check for non-specific binding of the various fluorochrome-conjugated antibodies to cells. Cells are incubated with the isotype control antibodies following the instructions above for preparing sample 1 and analyzed accordingly. The figure below shows a representative example of isotype control staining.
This antibody panel enables the simultaneous detection of both surface and intracellular markers for monitoring pluripotency. The figure below shows a representative result for the analysis of human induced pluripotent stem cells (iPS). The pluripotency markers SSEA-4, SSEA-5, TRA-1-60, Sox-2, and Oct 3/4 were expressed at high levels, whereas expression of the differentiation marker SSEA-1 (CD15) was low.
In contrast, cells differentiated towards the neural lineage expressed the pluripotency markers at low levels, or even shut off the expression of pluripotency markers, and up-regulated the differentiation marker SSEA-1 (CD15). Sox2 was expressed at high levels, as would be expected from neural precursors.
As a differentiation control, a second sample was stained with antibodies against PSA-NCAM and Pax6, which are markers for neuronal and neural progenitors, respectively. The large majority of cells (80%) co-expressed both markers.
PSC cultures were also monitored visually by light microscopy. The images show the typical morphology of PSC colonies (A in figure below) or cells differentiated into neural precursors (B in figure below).
Use the StemMACS™ Cryo-Brew for cryopreservation of pluripotent stem cells to ensure high viability and rapid recovery after thawing. To freeze as single cells, use Trypsin/EDTA for cell detachment. To freeze as cell clusters, use StemMACS™ Passaging Solution XF for detachment. Follow the protocol of the StemMACS Cryo-Brew data sheet.
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