Hema-3 Staining and Nuclear Morphology Analysis

Hema-3 staining reagents are purchased from Fisher Scientific Scientific (same as Diff-Quick reagents or Hema-Diff Stains sold by other suppliers).  There are 3 solutions in the kit.  

1. Solution 1 - Fixative (methanol).
2. Solution 2 - Xanthene
3. Solution 3 - Thiazine

This procedure can be used for cells attached to glass slides or on coverslips.  If your cells are initially in suspension, options for attaching to coverslips or slides include use of a Cytocentrifuge or plating cells onto a slides/coverslips coated with poly-lysine.  A cytocentrifuge has the advantage of slightly flattening the cells, which makes analysis of nuclear morphology easier. 

Cytocentrifuging neutrophils onto slides/coverslips.

1. We use a Shandon brand cytocentrifuge. Load ~ 50,000 neutrophils in about 300 ul to each funnel and spin 500 rpm 5 min to attach cells to the slide.  If you prefer cells on coverslips, place a coverslip (we use 12 mm round coverslips) on top of the slide under the cell funnel opening.

Hema-3 staining 

1. Aliquot some of each Hema-3 kit solution into small beakers or 50 ml conical tubes.  These aliquots can be reused for several weeks.
2. Dip the slide/coverslip with cells into the fixative for 10-15 sec. - Leaving cells in fixative for up to 10 min is OK.  Tip: If you have just spun cells onto coverslips or slides as above, it is better to gently pour the fixative over the cells as dipping the slide may cause some of the cells to detach before fixation is complete.
3. Drain excess fixative by touching the edge of the slide/coverslip on a Kimwipe.
4. Dip the slide/coverslip ~ 10 times in solution 2. Drain off excess.
5. Dip the slide/coverslip ~ 10 times in solution 3. Drain off excess.
6. NOTE:   Adjust the number of dips in each stain to adjust intensity if too dark or too light.
7. Dip several times in deionized water to rinse. Drain.
8. Let slides/coverslips air dry cell side up.
9. Attach coverslips to slides or mount coverslips over slides using Permount (Fisher).
10. Let dry over night at room temperature.
11. Take photos using a light microscope.  Tip:  We find that for phase contrast objectives the images of Hema-3 staining are often clearer if the phase ring and objective are mismatched (such as Phase 1 or Phase 3 ring and Phase 2 objective). One option is to try all the filters on the microscope turret to see which one is best.
12. For each experiment we typically generate 3-4 coverslips as technical replicates per condition and analyze at least 100 cells per coverslip and condition. Nuclear morphology/lobe counting is scored manually.

FYI - Silva-Del Toro is the correct last name of the first author.