Giemsa Staining for Colony Formation Assay: An In Vitro Staining Procedure to Assess Proliferative Capability of Mesenchymal Stem Cells

Samenvatting

Source: Schlaefli, P. et al. An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples. J. Vis. Exp. (2015)

In this video, we demonstrate the Giemsa staining procedure to assess the proliferative potential of mesenchymal stem cells to form viable colonies. Giemsa stain is a neutral stain primarily composed of azure and eosin dyes, which carry selective affinity for cellular components depending on their charge.

Protocol

1. Seeding for Expansion Cell Culture and CFU Plates

1. Resuspend the cells (consisting of erythrocytes and mononuclear cells) in 50 ml of basal medium: Alpha-MEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin and 2.5 mg/ml amphotericin B. Count the cells diluted 1:10 in Trypan Blue solution using a Neubauer chamber.
2. Plate the cells in cell culture flasks at the density of 5 x 10⁷ cells/cm² and incubate in a humidified incubator at 37 °C. If available, use hypoxic (5% oxygen) conditions. For the CFU assay control, plate 10⁹ cells in a cell culture dish of 10 cm diameter.
3. After 3 days of culture, mesenchymal stem cells are attached to the cell culture dish while other cells remain in suspension. Change the media with basal medium supplemented with 5 ng/ml basic fibroblast growth factor (bFGF). For the CFU assay control, treat the cell culture dish likewise.
4. Continue cell culture for a total of 2 weeks, exchanging media three times a week. If cells exceed 80% confluency, split by trypsinization. For the CFU assay control, exchange the media likewise, but do not split the cells for the course of the two weeks.

2. Giemsa Stain for CFU Assay

1. Prepare 10 ml of Giemsa-solution per dish: dilute the stock solution (7.6 g/l Giemsa in glycerol:methanol) 1:10 in sterile water (always prepare a fresh working dilution).
2. Remove the media from the cell culture dish and wash the cells with PBS. Be very precautious when applying liquid to the petri dish and avoid washing off the cells.
3. Fix cells in pure Methanol for 5 min at RT. Discard the Methanol. Add the Giemsa-solution and incubate for 60 min in a humidified incubator at 37 °C.
4. Wash two times with PBS. Air dry the plate head first on a paper towel. Count the colonies manually. This is best achieved using a marker pen on the back of the plate.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Basal Medium Components
PenStrep 100X	Gibco	15140122
Human FGF-basic	Peprotech	100-18B
MEM Alpha w/ Nucleoside, w/ stable Glutamine	Amimed	1-23S50-I
FBS Heat Inactivated	Amimed	2-01F36-I
Amphotericin B	Applichem	A1907
Generic
PBS	Applichem	964.91
Giemsa stain	Applichem	A0885
Consumables
Primaria cell cuture dish	Falcon	353803
Cell culture flask - Flask T300	TPP	90301
Equipment
Stripettes Serological Pipette 5ml	Corning	4487-200ea
Humidified incubator Heracells 240	Thermo scientific	51026331
Heraeus Multifuge 1S-R	Thermo scientific	75004331