Automated Sample Preparation Improves Stem Cell Populations...Automated Sample Preparation Improves...

Automated Sample Preparation ImprovesThroughput for Flow Cytometric Analysis of MouseStem Cell Populations

William L. Godfrey1, Li Liu2, Amy Yoder2, and Laura Pajak2

Beckman Coulter, Inc., 1Miami, FL, USA; 2Indianapolis, IN, USA

Application Information Bulletin: Stem Cell

Beckman Coulter Scientific Symposium

Automated Sample Preparation ImprovesThroughput for Flow Cytometric Analysis of MouseStem Cell Populations

ABSTRACT

Introduction. Although automation of sample preparation can provide significant labor savings and standardiza-tion, its application to flow cytometric studies has been hampered by the diversity and complexity of protocols. We demonstrate automation strategies using the Biomek* NXP automation workstation that provide well-resolved and reproducible detection of surface and intracellular markers on murine embryonic stem cells using both washed and no-wash protocols.

Methods. Mouse embryonic stem cells (Invitrogen) were cultured on gelatin-coated plates, either with or without differentiation factors, then harvested by trypsin and dispensed into 96-well plates for processing on a Biomek NXP system (Beckman Coulter) with on-board pipetting, mixing, and plate centrifuge components. Manual stains were done for comparison. Cells were fixed and permeabilized with 3 reagent systems (Beckman Coulter, Invitro-gen), and stained with titered conjugates against Nanog, OCT 3/4, SOX2, and myosin heavy chain. The on-board centrifuge was used for wash steps; one sample preparation system used a no-wash protocol. Samples were collected on an FC500 MPL* or a Gallios* cytometer and analyzed using Kaluza* software (Beckman Coulter).

Results. Multicolor expression of stem cell markers across populations was clearly demonstrated with all prepara-tion systems, including the expression of myosin heavy chain in differentiated cardiomyocytes. An automated washed cell assay using 96 wells required about 3.5 hours of preparation time, about the same time required to manually prepare 32 samples. When percent positive values were compared from 32 repetitions of the myosin heavy chain stain of the differentiated stem cell preparation containing cardiomyocytes, a 5.4% CV was obtained from automation and versus a 7.5% CV for manual preparation. The PerFix-nc* sample preparation system (Beck-man Coulter) provided good intracellular staining results without washing, greatly facilitating assay automation.

Conclusion. This work demonstrates that complex sample preparation work flows, including wash steps and intracellular staining, can be automated with relatively standard components and process steps. Automation can achieve preparation time savings with large numbers of samples while maintaining at least equivalent results and precision versus manual processing. As a result, it is possible to perform large flow cytometric staining stud-ies in an expandable manner with walk-away capability.

1S T E M C E L L • B E C K M A N C O U L T E R

MATERIALS & METHODS

Mouse embryonic stem (mES) cells (Invitrogen) were maintained in growth media containing LIF and 15% knockout serum replacement (KSR). For differentiation, cells were cultured in 15% FBS without LIF in a 384-well round-bottom polypropylene plate (Nunc) in 40 mL differentiation medium (various treatments for days 0 – 5). The embryoid bodies that formed by day 5 were transferred to gelatin-coated 96-well plates via Biomek NXP (Beckman Coulter) in 100 mL fresh media. After day 6, a portion of the adherent cells showed visible contrac-tion morphologies.

Cells were harvested on day 8 via Biomek FXP multichannel pipettor using Accumax (Millipore) and robust pipetting, then pooled for staining using a Biomek NXP with a Span-8 Pipettor. Conjugates used: myosin heavy chain-Alexa Fluor 488 (clone MF20), Nanog –Alexa Fluor 488 (clone eBioMLC-51), OCT3/4-PE (clone eBioEM92) and relevant isotype controls were from eBioscience (San Diego, CA); Sox2-Alexa Fluor 647 (clone 245610) and isotype control were from BD Bioscience (San Jose, CA). Sample preparation reagents: FIX&PERM (Invitrogen, Carlsbad, CA), IntraPrep and PerFix-nc (Beckman Coulter).

Samples were run on FC500 MPL or Gallios cytometers and the data were analyzed using Kaluza software (Beckman Coulter).

Staining index (SI) was calculated as:

(medianpositive – mediannegative)/(2 x StDevnegative).


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Figure 1. Sample Preparation Workflow Comparison


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RESULTS

Figure 2. Cardiomyocyte yield from full plate automation.

Cardiomyocyte yield from mES cells was assessed by % positive for myosin (using FIX&PERM) on a well-by-well basis, prepared in plate format by a Biomek NXP (n=96). The average cardiomyocyte yield is 44.5%, standard deviation 3.4%, coefficient of variation 7.7%. The data indicate that automated harvesting and sample preparation provides reproducible staining results.

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Figure 3. Comparison of car-diomyocyte staining between manual and automated sample preparation.

A mixed cell population of differentiated and undifferentiated mES cells were detached from plates on day 8 of differentiation and pooled, then distributed to 96 wells for automated sample preparation and into 32 wells for manual sample preparation, both using FIX&PERM. The throughput of automation using the Biomek NXP platform was higher than throughput of the manual process: 96 wells versus 32 wells in approximately 3.5 hours.

Figure 4. Cell Staining for imaging with the PerFix-nc kit.

PerFix-nc allows sample preparation without a wash step following fixation and permeabilization. The PerFix-nc kit was tested on undifferentiated and day 8 differentiated mES cells for staining. Mixtures of mES cells and day 8 differentiated mES cells (ratio 1:1) were detached and re-seeded into a 96 well imaging plate, then allowed 24 hrs to plate out. Media was aspirated and 45 μL PBS plus 5 μL PerFix-nc Reagent 1 was added for 15 min to fix the cells, followed by 50 μL PerFix-nc Reagent 2 with antibody conjugates for 30 minutes without washing. All liquid handling steps were performed on a Biomek 3000 platform. Sixteen wells required less than an hour to prepare for imaging. Images were taken using the 10X objective on an ImageXpress high content imager and processed with MetaXpress software (Molecular Devices). Cells were stained with either isotype controls (A) or with myosin heavy chain (green) and Nanog (blue). DAPI (red) was used to mark the locations of nuclei.

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Table 1. Comparison of flow cytometry staining results from the sample preparation protocols.

Staining results are shown for specimens prepared for flow cytometry using FIX&PERM and IntraPrep kits, as wells as automated and manual sample preparation with the PerFix-nc kit. Average percent positive is shown for Nanog, OCT3/4 and Sox2 staining, as well as CV and staining index (SI). N = 16 for each. See Figure 5 for example histograms.


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Figure 5. Flow cytometric histogram comparison for the sample preparation procedures.

Example histograms are shown for mES cells prepared and stained using the different sample preparation reagents: FIX&PERM (automated), IntraPrep (automated), and PerFix-nc (automated and manual). Staining used either isotype con-trols or antibody conjugates against early differentiation markers: Nanog-Alexa Fluor 488, OCT3/4-PE and Sox-Alexa Fluor 647. Staining index (SI) is calculated as described in Methods. PerFix-nc was used without wash steps, reducing sample preparation time and greatly facilitating automation.

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Figure 6. Flow cytometric dot plot comparison.

Dot plots are shown for mES samples prepared with the FIX&PERM (automated), IntraPrep (automated), and PerFix-nc (automated and manual) kits and stained with either isotype controls or antibody conjugates against early differentiation markers: Nanog-Alexa Fluor 488, OCT3/4-PE and Sox-Alexa Fluor 647.

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SUMMARY

• Automation improves throughput of stem cell staining while maintaining the precision and resolution of a manual process.

• The Biomek NXP with Span 8 pipettor provides efficient automation of 96-well plate-based staining.

• Automation solutions can facilitate the multiple repetitive studies often required for assay optimization.

• PerFix-nc provides a better resolution and less variabilty for intracellular staining for stem cell markers than the IntraPrep and FIX&PERM kits.

• PerFix-nc also provides easier automation capabilities with a no-wash protocol.

Gallios, IntraPrep and Kaluza are trademarks of Beckman Coulter, Inc. Beckman Coulter, Biomek and the stylized logo are registered

trademarks of Beckman Coulter, Inc and are registerd in the USPTO. Alexa Fluor is a registered trademark of Molecular Probes, Inc.

*PerFix-nc, Gallios and Kaluza are for Research Use Only. Biomek 3000, NXP and FXP are for Laboratory Use Only. Not for use in diagnostic

procedures.

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