Cell counting

11.10 Cell counting

11.10.1 Cell counting with a haemocytometer

For counting lymphocyte suspensions, use × 40 objective lens and count in the central, triple- ruled area of the haemocytometer (this area is used for red-cell counting in haematology).

Count the cells in the large triple-ruled squares (improved Neubauer ruling, Fig. 11.1) until a minimum of 100 unstained (viable) lymphocytes have been counted (see Fig. 11.2).

Calculation of the number of viable cells

Number viable lymphocytes/ml × 10 4

= Number lymphocytes counted

Number triple-ruled squares original dilution (if any) ×

C H A P T E R 1 1: Immunological manipulations in vivo

Fig. 11.1 The improved Neubauer ruling haemocytometer. For most applications in experimental immunology, the haemocytometer provides a cost-effective alternative to an electronic particle counter, unless you wish to use it for volume measurements. In principle, the number of cells in small volumes taken at random is counted to predetermined rules, and this value multiplied up to give an estimate of the total population. The figure shows the appearance of the central ruled area of the haemocytometer as it would

appear under a low-power lens on the light microscope. There are nine squares, each of 1 mm 2 ; as the depth of the counting chamber is 0.1 mm, cells settling into one of these squares have come from a volume of 0.1 mm 3 . If dealing with high-density cell populations, use a high-power lens (× 40) and count in the central triple-ruled areas (see Fig. 11.2); at low cell density count in one of the four peripheral, single-ruled areas. When counting lymphocyte suspensions, it is convenient to use a phase-contrast microscope and so distinguish viable (phase-bright) from dead (phase-dark) cells, as an adjunct to dye exclusion (see Section 11.10.2).

Fig. 11.2 Counting lymphocyte suspensions in a haemocytometer.

The figure shows an enlarged view of square 1 from Fig. 11.1. Cells falling across the top and left border lines of a square are considered to be in that square, whereas cells on the bottom and right borders are excluded. The diagram shows 25 cells (filled circles) in

an area of 1/25 mm 2 , depth of chamber 0.1 mm. To gain an acceptable degree of accuracy in this technique, you should count cells in the triple-ruled squares in the order shown in Fig. 11.1 until at least 100 cells have been included in the sample.

11.10CELL COUNTING

11.10.2 Dye exclusion test

This test is an extension of the method used to estimate cell viability (see Section 11.10.1).

MATERIALS Anti-lymphocyte serum (ALS) Rabbit anti-mouse immunoglobulin Guinea-pig serum (complement) Nigrosin dye Inbred mice, 4–6 weeks old

METHOD

1 Absorb the complement with mouse spleen and erythrocytes, approximately 0.1 ml packed cells/ml of serum, for 30 min at 4°C.

2 Centrifuge the absorbed complement. Use immediately or store at –20°C.

3 Prepare thymus and lymph-node suspensions from mouse donors. Estimate viability and

adjust to 5 × 10 7 viable lymphocytes/ml.

4 Prepare cell and serum mixtures according to the following Protocol, and incubate at 37°C for 30 min.

Protocol.

Tube number

1 : 40 1 : 80 1 : 160 1 : 320 1 : 640 final dilution

Anti-lymphocyte or normal rabbit serum

0.1 ml⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ Absorbed guinea-pig serum (1 : 5 initial dilution) 0.1 ml⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→

Lymph-node cells (5 × 10 7 /ml)

Repeat this Protocol using thymus cells with the same dilution of ALS and normal rabbit serum (NRS).

Assay 1 (see Protocol above)

Cells: lymph node. Antisera: ALS and preimmunization serum (NRS). Titrate NRS only to 1 : 40.

Assay 2 (see Protocol below)

Cells: lymph node. Antisera: anti-immunoglobulin (if assays 1 and 2 are performed simultaneously a second NRS control is not required).

5 After incubation stand the tubes in ice to prevent further complement fixation and cell lysis.

6 Count the number of viable cells in each suspension. Calculate the number of viable cells/ml and from this the percentage lysis for each tube according to the following equation:

% lysis = C N –C A × 100/C O

Continued

C H A P T E R 1 1: Immunological manipulations in vivo C H A P T E R 1 1: Immunological manipulations in vivo

Table 11.2 Protocol.

Tube number

Anti-immunoglobulin dilution

0.1 ml⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ Absorbed guinea-pig serum (1 : 5 initial dilution)

Lymph-node cells (5 × 10 7 /ml)

0.1 ml⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→

Repeat this Protocol using thymus cells with the same dilutions of anti-immunoglobulin.

8 Plot a graph of percentage lysis against antiserum dilution for each tissue and antiserum.

Interpretation of results • You should find that ALS kills all nucleated cells in the thymus and the lymph-node suspen-

sion to a high dilution of antiserum. This lack of T-cell specificity is, of course, to be expected as lymphocytes share many surface antigens.

• The percentage of cells killed by the anti-immunoglobulin serum should coincide with the expected percentage of B cells in the tissue used. • If the killing of thymocytes by anti-immunoglobulin or NRS exceeds 5%, there are probably

anti-species antibodies present, not related to the original immunizing procedure. In this case it is necessary to absorb all sera with liver membranes.

TECHNICAL NOTES • The sensitivity of this assay depends upon a high initial cell viability. Frequently 20–30% dead

cells are encountered in lymph-node suspensions. Dead cells may be removed as described in Section 6.4.

• The guinea-pig serum complement source must be absorbed with spleen and red cells before use

as it is sometimes itself cytotoxic. It was discovered that an agarose absorption may also be used to remove anti-mouse antibodies. Use 100 mg of agarose/ml of serum; absorb for 60 min at 4°C.

• In this assay it is advisable to count the number of viable cells after lysis rather than the number of dead cells, especially if centrifugation steps are included after killing. Dead cells are often broken up and lost during centrifugation.