May-Grunwald Giemsa Staining: A Method to Stain Bone Marrow Cells

Abstract

Source: Mopin, A. et al. A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model. J. Vis. Exp. (2016).

This video describes the technique of staining bone marrow cells using May- Grünwald Giemsa Staining. In the protocol, we will stain the murine myeloid leukemia cells, C149 cells, to analyze the morphology and differential counting.

Protocol

1. In vitro Characterization of the C1498 Cell Line

1. In vitro culture of C1498 cells
   1. Prepare complete RPMI (Roswell Park Memorial Institute) 1640 medium by adding 50 ml of fetal bovine serum (FBS), 5 ml of penicillin (100 U/ml)-streptomycin (100 µg/ml), 500 µl of 50 mM β-mercaptoethanol, 5 ml of N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), 5 ml of non-essential amino acids and 5 mL of sodium pyruvate to 500 ml of RPMI medium.
   2. Grow the C1498 cell line in complete RPMI. Harvest the cells in suspension by pipetting, and transfer the cells to a 50 ml tube. Centrifuge at 350 x....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C1498 cell line	ATCC	TIB 49
2-Mercaptoethanol, Gibco	ThermoFisher Scientific	21985
Fetal Bovine Serum (FBS)	ThermoFisher Scientific	10270
HEPES, Gibco (1 M)	ThermoFisher Scientific	15630
Penicillin-Streptomycin, Gibco	ThermoFisher Scientific	15140
RPMI 1640 Medium (Gibco, GlutaMAX Supplement)	ThermoFisher Scientific	61870
Trypan blue solution (0.4 %)	ThermoFisher Scientific	15250-061	concentration 1/2
Chamber & filter card (EZ Cytofunnel Shandon)	Thermo Scientific	A78710003
May-Grünwald solution	RAL Diagnostics	320070
Giemsa R solution	RAL Diagnostics	320310	concentration 1/20
pH 6.8 buffer solution	RAL Diagnostics	330368
Slides (Starfrost - ground edges 90)	Knittel Glass	VS1137# 077FKB
Microscope Cover Glasses, 24 x 24mm	Knittel Glass	VD1 2424 Y100
Shandon Cytospin 3 Cytocentrifuge	ThermoFisher Scientific