Evaluation of cells
11.6 Evaluation of cells
11.6.1 Morphology of thymus and blood leucocytes
Blood film
MATERIALS AND EQUIPMENT Mouse or human blood Microscope slides (clean overnight in acetone–ethanol 50 : 50 v/v) 95% methanol in water
METHOD
1 Grasp the mouse firmly by the nape of the neck using your thumb and forefinger. Hold the mouse’s tail between your little and third fingers with its abdomen towards you; use your middle finger to flex its spine slightly so it cannot struggle. This is a general position for immobilizing a mouse for intraperitoneal injection or, as here, cutting the tip from its tail with a pair of scissors. Humans are usually more cooperative; in this case a finger prick with
a sterile needle will draw sufficient blood.
2 Squeeze out 1 drop of blood and place it at one end of a clean glass slide.
3 Use a second, ‘spreader’ slide and touch the extreme edge of the drop of blood. Hold this slide at an angle of about 45 degrees.
4 Allow the blood to flow along the edge of the spreader slide and then push it away from the drop to obtain a film of cells (ideally in the shape of a bunsen flame).
5 Wave the slide in the air to dry it rapidly.
6 Fix in 95% methanol for 2 min.
With practice a serviceable, if not perfect, blood film can be produced. It is necessary to vary the size of the drop of blood and the amount taken up on the spreader slide to obtain an optimal dis- tribution of cells.
11.6.2 Smears of single-cell suspensions
Blood plasma is viscous and so protects the cells in whole blood from damage as they are smeared. Smears are more difficult to obtain from single-cell suspensions taken from organs.
Using a cytocentrifuge it is possible to obtain excellent preparations by simply suspending the cells in neat serum and smearing them as in the previous section.
11.6EVALUATION OF CELLS
MATERIALS AND EQUIPMENT Mouse Phosphate-buffered saline (PBS) Fetal bovine serum (FBS) Microscope slides (clean overnight in acetone–ethanol 50 : 50 v/v) 95% methanol in water
METHOD
1 Kill the mouse by cervical dislocation and remove the thymus into a Petri dish containing PBS or tissue culture medium.
2 Prepare a single-cell suspension.
3 Place 1 drop of the cell suspension on a clean slide and smear with a second slide.
4 Dry the slide in the air and fix in 95% methanol for 2 min.
5 Wash the slide in running tap water for 30 min to remove the FBS.
11.6.3 Cytocentrifuge technique
MATERIALS AND EQUIPMENT Mouse Phosphate-buffered saline (PBS) Fetal bovine serum (FBS) Microscope slides (clean overnight in acetone–ethanol 50 : 50 v/v) 95% methanol in water Cytocentrifuge
METHOD
1 Prepare a single-cell suspension from the mouse thymus as described in Section 6.3. After washing, finally resuspend in tissue culture medium with 10% FBS.
2 Load cytocentrifuge with glass slides and filter-paper strips.
3 Add 1 drop of cell suspension and 3 drops of tissue culture medium to each carrier block being used.
4 Centrifuge at 300 g for 10 min at room temperature.
5 Unload the glass slides, being careful to keep the slide and filter-paper strip together as you remove them from the carrier.
6 Remove the strip without smearing the cell preparation.
7 Dry the slide in the air and fix in 95% methanol for 2 min.
8 Wash the slides in running tap water for 30 min and then stain as required.
TECHNICAL NOTES • The concentration of the cells in the suspension and the volume used should be varied to obtain
the required cell density in the smear. • Human lymphocytes tend to be more fragile and should be centrifuged at 250 g.
C H A P T E R 1 1: Immunological manipulations in vivo
To examine the morphological details of the prepared cells, it is necessary to stain them. Pleasing results can be obtained with May–Grünwald/Giemsa staining.