Sep 07, 2022

Public workspacePluripotency markers staining

DOI
dx.doi.org/10.17504/protocols.io.b4yyqxxw
  • 1University of California, Berkeley;
  • 2Albert Einstein College of Medicine
Hanqin Li
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DOI: dx.doi.org/10.17504/protocols.io.b4yyqxxw
External link: https://doi.org/10.7554/eLife.79208
Protocol Citation: Hanqin Li, Dirk Hockemeyer, Frank Soldner 2022. Pluripotency markers staining. protocols.io https://dx.doi.org/10.17504/protocols.io.b4yyqxxw
Manuscript citation:
Hanqin Li, Oriol Busquets, Yogendra Verma, Khaja Mohieddin Syed, Nitzan Kutnowski, Gabriella R Pangilinan, Luke A Gilbert, Helen S Bateup, Donald C Rio, Dirk Hockemeyer, Frank Soldner (2022) Highly efficient generation of isogenic pluripotent stem cell models using prime editing eLife 11:e79208

https://doi.org/10.7554/eLife.79208
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 12, 2022
Last Modified: December 14, 2024
Protocol Integer ID: 58104
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000486
Abstract
This protocol describes the standard procedure for staining pluripotency markers, e.g. OCT4, SSEA4, alkaline phosphatase, and etc. on human pluripotent stem cells (hPSCs).

Protocol overview
A. Immunofluorescence staining
B. Alkaline phosphatase staining

General notes
Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
Materials

ItemVendorCatalog #
Para formaldehyde, PFA Sigma Millipore Sigma 158127
Bovine Serum Albumin (BSA) Sigma A4503
OCT4 primary antibody DSHB PCRP-POU5F1-1D2
SSEA4 primary antibody DSHB MC-813-70 (SSEA-4)
Donkey anti Mouse IgG (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor 594 Thermo Fisher A21203
NBT/BCIP substrate Sigma/Roche 11681451001
NBT/BCIP substrate Vector lab SK-5400
Triton-X100 Sigma X100
DAPI Sigma D9542
A. Immunofluorescence staining
A. Immunofluorescence staining
1h 50m
1h 50m
Wash cells once with PBS
Phosphate buffer saline, PBS, pH 7.4

AB
NaCl137 mM
KCl2.7 mM
Na2HPO410 mM
KH2PO41.8 mM
CaCl2•2H2O1 mM
MgCl2•6H2O0.5 mM

Fix cells with 4% PFA at TemperatureRoom temperature for Duration00:15:00

Note
You should not fix cells if staining for cell surface proteins

15m
4% PFA, pH 7.4
AB
PBS500 ml
PFA20 g
NaOHAdjust pH to 7.4
Final volume: 500 ml

Wash cells twice with PBS, incubate Duration00:05:00 in between washes at TemperatureRoom temperature

5m
Incubate in PBST for Duration00:30:00 at TemperatureRoom temperature to permeabilize cell membrane

30m
PBST, pH 7.4

AB
PBS1x
Triton-X1000.3%

Incubate in 3% Bovine Serum Albumin (BSA) for Duration01:00:00 at TemperatureRoom temperature

1h
3% Bovine Serum Albumin (BSA)
AB
PBS500 ml
BSA15 g

Incubate with primary antibody (1:200) in 3% BSA at Temperature4 °C DurationOvernight


1h
Wash three times with PBS, incubate Duration00:05:00 in between washes at TemperatureRoom temperature

5m
Incubate with secondary antibody (1:1,000) in 3% BSA at TemperatureRoom temperature for Duration01:00:00 in the dark and in a humidified chamber.

1h
Wash once with PBS
Incubate with 0.1 µg/ml DAPI for Duration00:05:00 at TemperatureRoom temperature

5m
Wash twice with PBS, incubate Duration00:05:00 in between washes at TemperatureRoom temperature

5m
Image cells, seal the plate with parafilm, and wrap with foil for longer storage at Temperature4 °C . Florescence is usually stable for several weeks.


B. Alkaline phosphatase staining
B. Alkaline phosphatase staining
20m
20m
Wash once with PBS
Fix cells with cold 4% PFA, Duration00:10:00 at TemperatureRoom temperature .

10m
Wash twice with PBS
Incubate cells with 100 mM Tris, pH 9.5, Duration00:10:00 at TemperatureRoom temperature

10m
100 mM Tris, pH 9.5
AB
Tris6.57g
HClAdjust pH to 9.5
Final volume: 500 ml

Add NBT/BCIP substrate in 100 mM Tris, and let reaction develop at TemperatureRoom temperature , avoiding light until color becomes clearly apparent in control cells.


Note
We have used NBT/BCIP substrate from two different vendors, which can be found in the materials list. The formulation for each are as follows:


ABC
NBT/BCIP substrate Vector1 drop of each reagent in 6 ml
NBT/BCIP substrate Roche100 µl to 10 ml


Wash in PBS and keep in PBS (violet product is soluble in organic solvent).