Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

Summary

Suspension immunocytochemical staining of human pluripotent stem cells (hPSCs) for cell-surface markers (SSEA-3/SSEA-4) was achieved based on use of a self-made cytospin apparatus to create a monolayer of cells for observation and quantification.

Abstract

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell sources due to their limitless self-renewal capabilities and their potential to differentiate into multiple cell types. The pluripotent state of hPSCs is typically assessed by techniques such as qPCR, immunocytochemistry, and by other in vitro and in vivo differentiation strategies into multiple cell types. Among these, immunocytochemical techniques have been developed for routine characterization of the undifferentiated state of hPSCs based on analysis of candidate intracellular and cell-surface biomarkers. Given the fact that hPSCs grow as colonies, problems arise in quantifying the expression of these markers at the individual cell level on a routine basis. Flow cytometry analyses serve to address this issue but require cell numbers and use of reagents that are not normally conducive for routine quality control assessment of hPSC cultures. Thus, the development of practical and reproducible means of creating monolayer cell samples with preserved integrity for marker evaluation has many advantages in stem cell research. This greatly benefits immunocytochemical analysis because individual cells from the monolayer can be easily observed and quantified for the expression of specific markers. Towards this goal, a self-made cytospin apparatus was constructed and optimized for use with immunocytochemical staining. Two cell-surface markers (SSEA3/SSEA4) expression were analyzed in a variant BG01 stem cell line for the purpose of this protocol.

Protocol

Part 1: Suspension Immunocytochemical Staining

1. Cells are harvested into a single cell suspension.
2. The cells are then transferred into 1.5-2mL microcentrifuge tubes and centrifuged at 200g for 4 minutes.
3. Cells are washed by aspirating out supernatant, re-suspended in 1mL of PBS-- (without Mg²⁺ and Ca²⁺) and then centrifuged again at 200g for 4 minutes. This should be done twice.
4. After washing, cells are then fixed by re-suspending them in 1mL of 4% paraformaldehyde (PFA) at room temperature for 10 minutes.
5. Cells are washed again twice with 1mL of PBS--.
6. After washing, block cells by re-suspending them in 1mL of Block Solution (6% serum/94% PBS--). Incubate these at room temperature for 45 minutes.
7. After, centrifuge tubes at 200g for 4 minutes and aspirate out Block Solution. Re-suspend cells in 1mL of the primary antibody solution (made with Block Solution at recommended dilution). Cells can either be incubated at room temperature for 1 hour or at 4°C overnight before proceeding to the next step.
8. Cells are then washed twice with 1mL of Block Solution.
9. After, re-suspend cells in 1mL of secondary antibody solution (made with Block Solution at recommended dilution) and incubate at room temperature for 1 hour.
10. After 1 hour, cells should then be washed again twice with 1mL of Block Solution.
11. After washing with Block Solution, wash cells again with 1mL of PBS--.
12. After the various washes, re-suspend cells in 1mL of DAPI nuclear stain (1:10,000 dilution of DAPI in PBS--) and incubate at room temperature for 5 minutes.
13. After 5 minutes, centrifuge tubes at 200g for 4 minutes. Aspirate out the DAPI nuclear stain solution and re-suspend cells in 1mL of PBS--.

Part 2: Incorporation of hPSCs in slides generated through cytospin apparatus

1. Plastic slides are prepared by making the desired amount of holes in them with a drill press. The diameter needs to be at most 1mm larger than the widest diameter of the micropipette tip(s) to be used.
2. Filter paper is cut to the parameters of the plastic slide with scissors.
3. Holes are then made in the filter paper. The size of the hole(s) should be large enough that the micropipette tip(s) fits snuggly through it.
4. After, one of the prepared plastic slides is placed on top and a glass slide in placed at the bottom of the cut filter paper (Figure 1). The layers are secured with tape.
5. A maximum of 100μL (depending on the desired monolayer density) of the stained cell suspension from Part 1 is then placed into the top of the micropipette tip(s).
6. If necessary, the apparatus with cell solution can be weighed and a counter-balance of similar weight created.
7. The cytospin apparatus and counter-balance can are then placed in a desktop centrifuge and centrifuged at 200g for 4 minutes.
8. After centrifugation, the cytospin apparatus is carefully disassembled in a way that doesn’t dislodge the cells from the glass slide.
9. If necessary, excess surrounding PBS-- on glass side in aspirated out.
10. Cells can then be viewed using a fluorescence microscope to check if the desired cell monolayer has been achieved.

Part 3: Mounting slides

1. A drop of the desired mounting media is placed directly in the center of each area containing cells.
2. After, a cover slip is gently lowered onto the slides trying to avoid air bubbles if possible.
3. Excess mounting media is then removed from slides.
4. After, the sides of the cover slips are sealed with nail varnish.
5. The slides can be kept in a dark storage area until results are observed and documented.

Discussion

For the purposes of our lab, a 35mm dish of stem cell cultures grown on mouse embryonic fibroblasts (MEFs) is allotted for the immunocytochemical staining procedure for use with the cytospin apparatus. The cells are then collected by means of enzymatic passaging, removing as much of the MEF layer as possible, into a single-cell suspension. If more effective stem cell isolation from the MEF layer is desired, colonies can be manually picked then digested into suspension. If feeder free conditions are used in growing the st...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lab Tek Permanox Chamber Slide	Thermo Fisher Scientific, Inc.	117437	Material for re-used plastic slides
Superfrost/Plus Microscope Slides Precleaned	Fisher Scientific	12-550-15
0.1-10μL Filter Tips	USA Scientific, Inc.	1121-3810
Microscope Cover Glass	Fisher Scientific	120542-B
Cytoseal 280	Richard-Allan Scientific	8311-4	Mounting Medium
DPBS	GIBCO, by Life Technologies	14190
Normal Goat Serum	Invitrogen	10000C
Mouse Anti-SSEA-4 Monoclonal Antibody	EMD Millipore	MAB4304	Primary Antibody for SSEA-4 staining
Rat Anti-SSEA-3 Monoclonal Antibody	EMD Millipore	MAB4303	Primary Antibody for SSEA-3 staining
Alexa Fluor 488 Goat Anti-Mouse IgG	Invitrogen	A11029	Secondary Antibody for SSEA-4 staining
Alexa Fluor 488 Labeled Goat Anti-Rat IgG	Invitrogen	A11006	Secondary Antibody for SSEA-3 staining
DAPI, Dihydrochloride	Calbiochem	268298