! CENTER FOR REGENERATIVE MEDICINE of BOSTON UNIVERSITY and BOSTON MEDICAL CENTER ! nd 670 Albany Street, 2 Floor Boston, MA 02118 Tel: 617-414-2963 http://www.bumc.bu.edu/stemcells/

iPSC CORE !

Immunostaining of hiPS Cells for Pluripotency Markers

For phenotypic assessment of the differentiation status of hES/hiPS cells, mature cell colonies are stained for cell-surface-antigen (SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81) expression using immunofluorescence techniques.

Materials and Reagents:

• ES Characterization Kit (EMD Millipore Cat# SCR001) • 16% Paraformaldehyde (PFA) (EMS Cat# 15710) • Wash Buffer (1X TBST: TBS NaCl, pH 7.4; Tween-20; Boston Bioproducts 1BB-181) • DPBS +/+ (Life Tech. Cat# 14040-133) • Normal goat serum (Vector, Cat# S-1000) • TritonTM X-100 (Sigma, Cat# T8787) • Alexa FluorTM goat α-mouse AF488 or AF546 (Life Tech. Cat# A21042; A11003) • DAPI stain (Invitrogen D1306) (stock: 5 mg/mL in H20) • 12-well plates (Fisher Cat# 07-200-84)

1. Human iPS cells are plated onto feeder layers or Matrigel in 12-well plates and grown for at least 5 days prior to fixation. Most of the colonies in the well should be medium size at the time of fixation.

2. To fix hiPS cells, aspirate off culture media and add 1 mL 4% paraformaldehyde/DPBS for 20 minutes at room temperature (RT). Transfer spent fixative to appropriate waste container for proper disposal.

3. Wash 3x (5 min each) with 1 mL Wash Buffer.

4. Permeabilize cells with 1 mL 0.1% Triton X-100/DPBS for 10 minutes at RT.

5. Wash 3x (5 minutes each) with 1 mL Wash Buffer.

6. Add 1 mL Blocking Solution (4% normal goat serum/DPBS) for 30 minutes at RT. Do not rinse wells with Wash Buffer prior to adding primary antibodies.

7. Dilute primary antibodies to working concentrations (see below) in Blocking Solution. Add 0.5 mL primary antibody in Blocking Solution to each well and incubate for 1 hour at RT.

Primary antibodies Working concentration Secondary antibodies TRA-1-60 IgM mAb 1:50 AF488 gam (A21042, Life Tech) 1:250 TRA-1-81 IgM mAb 1:50 AF488 gam (A21042, Life Tech) 1:250 SSEA-1 IgM mAb (mouse) 1:50 AF488 gam (A21042, Life Tech) 1:250 SSEA-4 IgG mAb 1:50 AF546 gam (A11003, Life Tech) 1:100

8. Wash 3x (5 min each) with Wash Buffer.

670 Albany Street, 2nd Floor. Boston, MA 02118. 617-414-2969. CENTER! FOR REGENERATIVE MEDICINE of BOSTON UNIVERSITY and BOSTON MEDICAL CENTER

9. Dilute secondary antibodies (Fluorophore-labeled goat anti-mouse IgM or anti-IgG, appropriate to the isotype of the primary antibody) in DPBS (1:100-1:250) just prior to use (see above). Add 0.5 mL secondary antibody to each well and incubate for 30 min to 1 hour at RT. Cover with aluminum foil.

10. Wash 3x (5 minutes each) with Wash Buffer.

11. Dilute DAPI stock (5 mg/mL) to 1:2000 in DPBS just prior to use. Add 0.5 mL diluted DAPI stain to each well and incubate at RT for 5 min. Aspirate and perform quick rinse with 1 mL DPBS. Cover plate in aluminum foil and store at 4oC in DPBS.

12. Images can be visualized using a fluorescence microscope. For recored keeping, obtain phase/brightfield and immunofluorescent images using 4x and/or 10x objectives.

Reference: “ES Cell Characterization Kit Manual” (Millipore, Cat# SCR001).

670 Albany Street, 2nd Floor. Boston, MA 02118. 617-414-2969. CENTER! FOR REGENERATIVE MEDICINE of BOSTON UNIVERSITY and BOSTON MEDICAL CENTER

2016-03-01 Drafted by MFJ MFJ

670 Albany Street, 2nd Floor. Boston, MA 02118. 617-414-2969.