Multicolor flow cytometry analysis of human pluripotent stem cell cultures

Transcription

1 Multicolor flow cytometry analysis of human pluripotent stem cell cultures ackground Thorough characterization of pluripotency is mandatory during the establishment of newly derived human embryonic or induced pluripotent stem cell (hipsc) lines. However, it is equally important to constantly monitor established cell lines in order to detect signs of spontaneous differentiation early on. Monitoring of the pluripotency and differentiation status of PSC cultures is commonly done by immunofluorescence microscopy analysis. This method, however, is laborious and does not allow for reliable quantification of cell populations. Despite its vast potential for sophisticated and detailed cell analysis, flow cytometry is used only seldom in stem cell research. Here we present a multicolor flow cytometry protocol that allows for the simultaneous quantification of intracellular and surface markers for easy monitoring of the pluripotency status of human PSC cultures. Material Antibodies Table specifies the antibodies used for the multicolor panel and the corresponding isotype control antibodies. The recommended dilution for each antibody is specified in the datasheet. uffers and reagents uffer consisting of PS, ph 7.2,.5% SA, 2 mm EDTA is prepared by diluting MACS SA Stock Solution (# ) :2 with automacs Rinsing Solution (# ). Keep the buffer cold (2 8 C). Inside Stain Kit (# ) for intracellular staining MACS Comp ead Kit, anti-rea (#34-693) to set up the PE-Vio 77 channel for detection of the differentiation marker SSEA Antibody Clone Order no. Detection antibodies Anti-TRA-6-PE, human REA Anti-SSEA-PE-Vio 77, human and mouse REA , human REA Anti-SSEA-5-Violue, human 8e , human and mouse REA , human and mouse REA Isotype control antibodies Mouse IgG-Violue IS5-2F REA Control (I)-FITC REA REA Control (I)-APC REA REA Control (S)-PE REA REA Control (S)-VioGreen REA REA Control (S)-PE-Vio 77 REA Table : Multicolor panel and isotype controls for flow cytometry of PSCs and differentiated precursor cells. Cells Human PSCs were cultured in StemMACS ips-rew XF, human (#34-368), in 6-well plates coated with Matrigel hesc-qualified Matrix. To confirm that the flow cytometry assay reliably detects a loss of pluripotency, we also analyzed hipscs differentiated towards the neural lineage. oth pluripotent and differentiated cells were treated with.5% trypsin/edta to obtain single cells for flow cytometry analysis. Multicolor flow cytometry analysis of hpscs April 28 /5

2 Flow cytometry Cell staining The panel for analysis of PSCs 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, e.g., fixed and permeabilized, in the same way as the stained samples. Please see the pipetting scheme in table 2 for an overview. As SSEA is not expressed in pluripotent cells, the MACS Comp ead Kit, anti-rea is used for compensation of PE-Vio 77 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.. Determine cell number. Use ⁶ ips cells per sample. 2. Centrifuge cell suspension at 3 g for 5 minutes. Aspirate supernatant completely. 3. Resuspend sample in 77.8 μl of buffer. This will be the cell sample stained with the complete panel. 4. Resuspend samples 2 7 in µl of buffer. This will be the unstained and single-stained samples. 5. Add µl of each of the following antibodies, detecting the surface markers, to sample : i) Anti-TRA-6-PE, ii), and iii) Anti-SSEA-5-Violue. Add 2.2 µl of Anti-SSEA-PE-Vio Add µl of one of the antibodies detecting a surface marker to samples Do not add antibody to samples 2, 6, and 7 at this point. 8. Mix well and incubate for minutes in the dark in the refrigerator (2 8 C). Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times. 9. Wash cells by adding ml of buffer per sample and centrifuge at 3 g for 5 minutes. Aspirate supernatant completely.. Resuspend all samples in µl of buffer. Add µl of Inside Fix per sample.. Mix well and incubate for 2 minutes in the dark at room temperature. 2. Wash cells by adding ml of buffer and centrifuge at 3 g for 5 minutes. Aspirate supernatant completely. 3. Resuspend sample in 9 μl of Inside Perm. 4. Resuspend samples 2 7 in μl of Inside Perm. 5. Add µl of each of the following antibodies, detecting intracellular markers, to sample : i), and ii). 6. Add µl of one of the antibodies for intracellular markers to samples 6 and Mix well and incubate for 5 minutes in the dark at room temperature. 8. Wash cells by adding ml of Inside Perm and centrifuge at 3 g for 5 minutes. Aspirate supernatant completely. 9. Resuspend cell pellet in a suitable amount of buffer for analysis by flow cytometry. A volume of ml of buffer per sample is recommended. Note: Fixed and permeabilized cells are smaller than viable cells. Thus, FSC/SSC settings of the flow cytometer might have to be adjusted. Cell surface staining Intracellular staining Sample uffer Anti-TRA- -6-PE Anti-SSEA- PE-Vio 77 Anti-SSEA-4- VioGreen Anti-SSEA-5- Violue Inside Perm Anti-Sox2- FITC Anti-Oct3/4 Isoform A-APC. Multicolor panel 77.8 µl µl 2.2 µl µl µl 9 µl µl µl 2. Unstained sample µl µl 3. Single-color staining µl µl µl 4. Single-color staining µl µl µl 5. Single-color staining µl µl µl 6. Single-color staining µl µl µl 7. Single-color staining µl µl µl Table 2: Pipetting scheme for surface and intracellular staining. Cell surface staining refers to steps 3 8 of the protocol. Intracellular staining refers to steps 4 9. Multicolor flow cytometry analysis of hpscs April 28 2/5

3 Instrument setup. Set up the scatter and voltages for all channels with the unstained cell sample (sample 2) (fig. A). Specify the trigger. 2. Use single-color stainings (samples 3 7) to define the compensation (fig. ). 3. To set up the PE-Vio 77 channel, temporarily lower the trigger to detect all the Comp eads and define the compensation (fig. C). Afterwards change the trigger settings back to the value specified in step. A Side scatter Anti-TRA-6-PE C Side scatter ² ¹ FSC/SSC 64,9% Forward scatter 78,28% 2,27%,45%,% ¹ ² ² ¹ Uncompensated FSC/SSC 99,92% Forward scatter Uncompensated Anti-SSEA-PE-Vio 77 98,87%,8% ¹ ² Figure : Setting up the flow cytometer for compensation of spectral overlap. (A) Adjustment of scatter and voltage using an unstained sample. PE vs. FITC is shown as an example. () Compensation using a PE-stained cell sample; PE vs. FITC is shown as an example. (C) Compensation of the PE-Vio 77 channel using MACS Comp ead Kit Anti-REA according to the accompanying protocol. PE-Vio 77 vs. PE is shown as an example. Anti-TRA-6-PE ² ¹ ² ¹,67%,28% 99,23%,29%,47%,% ¹ ² ² ¹,67%,28% 98,87%,8% ¹ ² PE-Vio 77-A ¹ ² ¹ ² ² ¹ Compensated Compensated Anti-SSEA-PE-Vio 77 Isotype controls 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 as indicated for sample and analyzed accordingly. Figure 2 shows a representative example of isotype control staining. ¹ ² REA Control (I)-FITC ¹ ² REA Control (S)-PE-Vio 77 ¹ ² ¹ ² REA Control (S)-PE Figure 2: Isotype control staining. Cells were incubated with the various isotype control antibodies (red line) or left unstained (black line) and analyzed by flow cytometry on the MACSQuant Analyzer. Results REA Control (S)-VioGreen ¹ ² REA Control (I)-APC ¹ ² Mouse IgG-Violue The antibody panel described here enables the detection of both surface and intracellular markers for monitoring pluripotency. Figure 3A shows a representative result for the analysis of human ipscs. The pluripotency markers SSEA-4, SSEA-5, TRA-6, Sox-2, and Oct 3/4 were expressed at high levels, whereas expression of the differentiation marker SSEA 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 (fig. 3). Sox2 was expressed at high levels, as could be expected from neural precursors (fig. 3). As a differentiation control, we stained a second sample with antibodies against PSA-NCAM and Pax6, which are markers for neuronal and neural progenitors, respectively. The large majority of cells (8%) co-expressed both markers (fig. 3). Multicolor flow cytometry analysis of hpscs April 28 3/5

4 A ² ¹,67%,28% 98,87%,8% ¹ ² Anti-SSEA-5-Violue ² ¹,8% 99,24%,24%,34% ¹ ² ² ¹,3% 98,%,2%,5% ¹ ² ² ¹,3% 99,6%,24%,29% ¹ ² Anti-TRA-6-PE Anti-TRA-6-PE Anti-TRA-6-PE Anti-TRA-6-PE ² ¹,85% 98,58%,38%,9% ¹ ² ² ¹,4% 98,99%,27%,34% ¹ ² Anti-SSEA-PE-Vio 77 ² ¹,3% 5,9%,77% 84,2% ¹ ² Anti-TRA-6-PE Anti-TRA-6-PE Anti-TRA-6-PE SSEA SSEA-5 SSEA-4 TRA-6 Sox2 Oct 3/4 Oct 3/ Sox TRA SSEA SSEA SSEA ,46%,8% ² ¹ 66,24%,5% ¹ ² Anti-SSEA-5-Violue ² ¹,67%,28% 98,87%,8% ¹ ² ² ¹,67%,28% 98,87%,8% ¹ ² ² ¹ 6,6%,27% 92,74%,39% ¹ ² Anti-TRA-6-PE Anti-TRA-6-PE Anti-TRA-6-PE Anti-TRA-6-PE ² ¹,37% 5,53% 2,49% 9,6% ¹ ² ² ¹,67%,28% 98,87%,8% ¹ ² Anti-SSEA-PE-Vio 77 ² ¹ 4,35%,49% 58,85%,3% ¹ ² Anti-Pax6-PE ² ¹ 8,94% 79,65% 5,8% 5,6% ¹ ² Anti-SSEA-5-Violue Anti-TRA-6-PE Anti-PSA-NCAM-APC SSEA SSEA-5 SSEA-4 TRA-6 Sox2 Oct 3/4 Oct 3/ Sox TRA SSEA SSEA SSEA low marker expression high Figure 3: Multicolor flow cytometry analysis of undifferentiated (A) and differentiated () human ipscs. Cells were stained with the antibodies as indicated and analyzed by flow cytometry on the MACSQuant Analyzer. Unstained cells were used as a control for gating. Numbers in the heat map specify percentages of single-positive (bold numbers) and double-positive cells. Multicolor flow cytometry analysis of hpscs April 28 4/5

5 PSC cultures were also monitored visually by light microscopy. The images show the typical morphology of PSC colonies (fig. 4A) or cells differentiated into neural precursors (fig. 4). A Conclusion The antibody panel described in this application note enables reliable monitoring of the pluripotency status of PSCs by multicolor flow cytometry. The procedure is fast and easy to perform for routine monitoring. Intracellular and surface markers are detected simultaneously. Figure 4: Cultures of undifferentiated human ipscs (A) and ipscs differentiated towards the neural lineage (). Shown are light microscopy images of the same cultures that were used for flow cytometry. Miltenyi iotec GmbH Friedrich-Ebert-Straße ergisch Gladbach Germany Phone Fax macs@miltenyibiotec.de Miltenyi iotec provides products and services worldwide. Visit to find your nearest Miltenyi iotec contact. Unless otherwise specifically indicated, Miltenyi iotec products and services are for research use only and not for therapeutic or diagnostic use. automacs, MACS, MACSQuant, REAfinity, Vio, Violue, and VioGreen are registered trademarks or trademarks of Miltenyi iotec GmbH and/or its affiliates in various countries worldwide. Copyright 28 Miltenyi iotec GmbH and/or its affiliates. All rights reserved. Multicolor flow cytometry analysis of hpscs April 28 5/5