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

Protocolo

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 g for 10 min, and remove the supernatant.
   3. Add 20 ml of phosphate-buffered saline (PBS) (1x) solution, centrifuge at 350 x g for 10 min, and remove the supernatant.
   4. Resuspend the cells in 10 ml of Fluorescence-Activated Cell Sorter (FACs) buffer (2.5 g of bovine serum albumin (BSA) powder and 2 ml of 0.5 M ethylenediaminetetraacetic acid (EDTA) solution in 500 ml of PBS solution). Count the cells using a Thoma cell counting chamber after staining the cells with trypan blue.

2. Preparation of a cell suspension on slides for microscopy

1. Wash 10⁶ of the harvested C1498 cells (obtained in step 1.1.4) with 5 ml of cold FACs buffer twice, and dilute the cells in 1 ml of cold FACs buffer. Place the tubes on ice.
2. Place slides into disposable chambers with pre-attached filter cards and place these into a cytocentrifuge.
3. Add 100 µl of FACs buffer to each chamber and filter card, and spin them for 2 min at 4.52 x g.
4. Add 100 µl of cells to each chamber and filter card, and spin the cells at 4.52 x g for 2 min.
5. Carefully remove the slides from the chambers and air-dry the slides before staining them with May-Grünwald Giemsa stain.

3. May-Grünwald Giemsa (MGG) staining

1. Stain the cells (prepared in section 2) by immersing the slides into a Coplin jar containing May-Grünwald solution for 3 min.
2. Transfer the slides into a Coplin jar containing the pH 6.8 buffer solution for 1 min.
3. Stain the slides by placing them into a Coplin jar containing the Giemsa R solution (diluted to 1/20 in pH 6.8 buffer solution) for 10 min. Wash the slides with neutral water (pH 7) for 10 sec.
4. Drain and air-dry the slides. Mount the slides by applying one drop of mounting medium 2 onto the cells. Place one edge of the cover-glass onto the slide and carefully lower it onto the cells using forceps. Press gently on the cover-glass to remove any air bubbles.

Materiais

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