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1. In vitro Characterization of the C1498 Cell Line
- In vitro culture of C1498 cells
- 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.
- 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.
- Add 20 ml of phosphate-buffered saline (PBS) (1x) solution, centrifuge at 350 x g for 10 min, and remove the supernatant.
- 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
- 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.
- Place slides into disposable chambers with pre-attached filter cards and place these into a cytocentrifuge.
- Add 100 µl of FACs buffer to each chamber and filter card, and spin them for 2 min at 4.52 x g.
- Add 100 µl of cells to each chamber and filter card, and spin the cells at 4.52 x g for 2 min.
- 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
- Stain the cells (prepared in section 2) by immersing the slides into a Coplin jar containing May-Grünwald solution for 3 min.
- Transfer the slides into a Coplin jar containing the pH 6.8 buffer solution for 1 min.
- 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.
- 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.