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.
Part 1: Suspension Immunocytochemical Staining
Part 2: Incorporation of hPSCs in slides generated through cytospin apparatus
Part 3: Mounting slides
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 stem cells cultures, the colonies can simply be scraped off and digested into suspension. While an initial single-cell suspension is ideal, any cellular clumps should effectively be broken apart throughout the numerous washing and re-suspension steps called for in the immunocytochemical staining procedure.
The video focused on the use of extracellular markers for staining hPSCs. If intracellular staining of the hPSCs is desired, an additional step before the addition of Block Solution (Step 1.6) needs to be done wherein the cells are washed in 1mL of Permeabilization Solution (50mLs of High Salt Buffer with 25μL of Tween 20) three times before re-suspending them in Block Solution. Another critical point that should be mentioned is that from Step 1.9 in the procedure, care should be taken in minimizing light exposure to the samples to prevent fluorescence bleaching of the secondary antibody and DAPI nuclear stain.
Because of the numerous washing and re-suspension steps in the procedure, an entire 35mm dish with a good amount of passage-ready stem cell colonies, estimated to have around 400k-600k cells, is recommended to be digested and used in the initial suspension. This is done in anticipation of some cell loss due to the nature of the procedure. Theoretically, a much smaller amount of cells can be used in the initial suspension but an extra amount of care must be taken in order to further minimize cell loss. When loading onto the cytospin apparatus after the immunocytochemical staining procedure, a cell density of 10k-50k is ideal. It should be mentioned that, while 100μL is the maximum amount that can be loaded onto a cytopsin apparatus that uses a 0.1μL – 10μL micropipette tip, a smaller amount (around 20μL – 30μL) containing the ideal cell density is recommended. This minimizes unnecessary overflow of liquid onto the glass slide. Finally, when using the cytospin apparatus with the centrifuge, as long as the apparatus is secure from lateral movement, the force created by the centrifuge while running has, to our knowledge, been sufficient in keeping the apparatus from flipping or falling off.
Ingredients for solutions used in the immunocytochemical procedure:
The authors have nothing to disclose.
Partial funding for this work was provided by NSF-CAREER 0744556 (Rao) and a scholarship through the VCU-HHMI Science Education and Research Program (Pascual).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Lab Tek Permanox Chamber Slide | Thermo Fisher Scientific | 117437 | Material for re-used plastic slides | |
Superfrost/Plus Microscope Slides Precleaned | Fisher Scientific | 12-550-15 | ||
0.1-10μL Filter Tips | USA Scientific | 1121-3810 | ||
Microscope Cover Glass | Fisher Scientific | 120542-B | ||
Cytoseal 280 | Richard-Allan Scientific | 8311-4 | Mounting Medium | |
DPBS | Gibco | 14190 | ||
Normal Goat Serum | Invitrogen | 10000C | ||
Mouse Anti-SSEA-4 Monoclonal Antibody | Millipore/Chemicon | MAB4304 | Primary Antibody for SSEA-4 staining | |
Rat Anti-SSEA-3 Monoclonal Antibody | Millipore/Chemicon | MAB4303 | Primary Antibody for SSEA-3 staining | |
Alexa Fluor 488 Goat Anti-Mouse IgG | Invitrogen/ Molecular Probes | A11029 | Secondary Antibody for SSEA-4 staining | |
Alexa Fluor 488 Labeled Goat Anti-Rat IgG | Invitrogen/ Molecular Probes | A11006 | Secondary Antibody for SSEA-3 staining | |
DAPI, Dihydrochloride | CalBioChem | 268298 |