Giemsa Staining for Colony Formation Assay: An In Vitro Staining Procedure to Assess Proliferative Capability of Mesenchymal Stem Cells
Published: April 30, 2023
Abstract
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 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. Plate the cells in cell culture flasks at the density of 5 x 10⁷ cells/cm² and incubate in a humidified incubator a…
Declarações
The authors have nothing to disclose.
Materials
| 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 |
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Giemsa Staining for Colony Formation Assay: An In Vitro Staining Procedure to Assess Proliferative Capability of Mesenchymal Stem Cells. J. Vis. Exp. (Pending Publication), e21050, doi: (2023).