This panel has been tested on foreskin fibroblasts reprogrammed in a feeder-free system using a small molecule cocktail, SMC4 (Please see Valamehr et al, 2012 for details).Using the panel, cells were isolated using Fluorescence Activated Cell Sorting (FACS) at an early stage, approximately days 20 post-transduction. Cells that were bulk sorted at this stage were further grown. At approximately 30 days post transduction, cells were both bulk and single sorted. As cells, reprogramming methods, and time courses can differ, specific sorting, analysis times, and results may vary.

Directions for Cell Analysis

(1) Detach cells of interest from the culture dish. Investigators are encouraged to detach cells at 37°C using Accutase™ Cell Detachment Solution (Cat. No. 561527).Mild to moderate triturating of the cell suspension is recommended to achieve a single cell suspension.

(2) Use media or 1XPBS to remove residual cells from plate.If clumps are present, cells can be filtered using a 70 mm BD Falcon™ cell strainer (Cat. No. 352350).

(3) Collect and spin down cells.

(4) Resuspend at 5 - 10 million cells/ml in BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656). Alternatively, a solution containing 1 x PBS,1% FCS, and 0.09% sodium azide may be used.

(5) Label tubes and add the corresponding antibody conjugates as described below. Vortex CompBead Plus prior to use:

Tube LabelAdd (1 test or drop)

1. Unlabeled compensation Negative CompBead plus + Anti-mouse Compbead plus

2. PE compensation Negative CompBead plus + Anti-mouse Compbead plus + TRA-1-60 PE

3. PerCP-Cy™5.5 compensation Negative CompBead plus + Anti-mouse Compbead plus + CD13 PerCP-Cy™5.5

4. Alexa Fluor® 647 compensationNegative CompBead plus + Anti-mouse Compbead plus + SSEA-4 Alexa Fluor® 647

4. Cells alone Nothing

5. Isotype control Ms IgGM PE + Ms IgG1 PerCP-Cy5.5 + Ms IgG3 Alexa Fluor® 647

6. Analysis sample TRA-1-60 PE + CD13 PerCP-Cy5.5 + SSEA-4 Alexa Fluor® 647

(6) Add 100 μl cells into appropriately labeled 12 x 75mm polystyrene tubes.

(7) Incubate in the dark for 30 minutes.

(8) Wash twice with 2 ml BD Pharmingen™ Stain Buffer (FBS).

(9) Resuspend sample in appropriate volume (350-400μl) of BD Pharmingen™ Stain Buffer (FBS) to run on a flow cytometer.

Directions for Cell Sorting

(All steps performed using sterile techniques)

(1) Detach cells of interest from the culture dish. Investigators are encouraged to detach cells at 37°C using Accutase™ Cell Detachment Solution (Cat. No. 561527).Mild to moderate triturating of the cell suspension is recommended to achieve a single cell suspension.

(2) Use media to remove residual cells from plate.If clumps are present, cells can be filtered using a 70 mm BD Falcon™ cell strainer (Cat. No. 352350).

(3) Collect and spin down cells.

(4) Resuspend cells at around 5-10 million cells/ml in appropriate sorting buffer. For this application, we utilized HBSS/4%FBS/10mM HEPES/1X Pen/Strep/10uM Thiazovivin.

(5) Use sterile 12x75mm tubes with caps. Label tubes and add the corresponding antibody conjugates as described below:

Tube LabelAdd (1 test or drop)

1. Unlabeled compensation Negative CompBead plus + Anti-mouse Compbead plus

2. PE compensation Negative CompBead plus + Anti-mouse Compbead plus + TRA-1-60 PE

3. PerCP-Cy™5.5 compensation Negative CompBead plus + Anti-mouse Compbead plus + CD13 PerCP-Cy™5.5

4. Alexa Fluor® 647 compensationNegative CompBead plus + Anti-mouse Compbead plus + SSEA-4 Alexa Fluor® 647

4. Cells alone Nothing

5. Isotype control Ms IgGM PE + Ms IgG1 PerCP-Cy5.5 + Ms IgG3 Alexa Fluor® 647

6. Sort sample 2 tests (10ul) each of TRA-1-60 PE + CD13 PerCP-Cy5.5 + SSEA-4Alexa Fluor® 647

(6) Add 50-100 ul of cells to tube 4 and 5.

(7) Add up to 500ul cells (5 million cells) of cells to tube 6.

(8) If you wish to sort more than 5 million cells we recommend replicating tube 6 with additional cells that you wish to sort.

(9) Incubate cells on ice in the dark for 20-30 minutes.

(10) Wash cells once 2ml in appropriate sorting buffer (tubes 4-6). Compensation tubes 1-4 can be washed with 1X sterile PBS.

(11) Resuspend cells in appropriate sorting buffer (we utilized HBSS/4%FBS/10mM HEPES/1X Pen/Strep/10uM Thiazovivin) at a concentration of 2.5 to 5 million cells/ml.Alternatively please contact your cell sorter operator to get a suggested final concentration of cells for sorting

Kit Considerations:

Choosing a Cell Detachment Enzyme: Investigators are encouraged to use Accutase™ Cell Detachment Solution (Cat. No. 561527), as cell death with this detachment method has been observed to be minimal.

Bulk and Single Cell Fluorescence Activated Cell Sorting Considerations: Using the panel, cells were bulk sorted at early and late reprogramming stages (approximately days 20 and 30 post transduction) and single cell sorted at approximately day 30 post-transduction.

For additional details on workflow and the concept of single cell sorting of hiPSC please see Valamehr et al, 2012 for details. For single cell sorting, it is recommended to plate cells into 96-well plates at various cell numbers (we have been successful with cells plated at 1, 3, and 9 cells per well) since plating efficiencies can vary.

Note that in cells reprogrammed in and grown in SMC4 media, Fibronectin (Cat. No. 356008) used at 5 µg/ml can be beneficial for both bulk and single cell sorting when kept in the media approximately 48 hours post-sort. Additionally, if cells that are plated in 96-well plates need to be grown on the same matrix for more than one week, fibronectin may be added at 5 µg/ml to assist in maintenance of cell attachment.

Data Analysis: A cluster based gating approach is recommended when analyzing data.