Autoradiographic labelling of lymphocytes

8.2.3 Autoradiographic labelling of lymphocytes

The investigation of the surface components of lymphocytes using fluorochrome-labelled antibodies gives increased sensitivity over that achieved using a radioactive isotope.

Autoradiography is semiquantitative, where the relative number of grains per cell is depend- ent upon the number of surface determinants detected. The relative distribution of the determin- ants throughout the cell population may be estimated and, within the same experiment, the

8.2LYMPHOCYTE SURFACE MEMBRANE

Fig. 8.3 Anti-light-chain (anti-LC) labelling of chicken bursa cells.

Cells: 1-day-old white leghorn bursa; antiserum: 125

I anti-LC antibodies; control: 125 I-anti-keyhole limpet haemocyanin antibodies.

relative concentration and distribution of determinants between two cell populations. For instance, it has been shown that B cells vary widely in the number of available immunoglobulin molecules on their surface (Fig. 8.3) and that cells differ quantitatively in their membrane con- tent of immunoglobulin. The basic autoradiographic method as described below may be used in any situation where a radioactive isotope is introduced into or onto a cell or tissue.

Example: labelling of chicken lymphocytes

MATERIALS AND EQUIPMENT 1-day-old chickens (see also Section 11.9) Anti-immunoglobulin antibody: either purified by acid elution, or an IgG fraction Control: either an irrelevant purified antibody, e.g. anti-keyhole limpet haemocyanin (anti-KLH),

or normal rabbit IgG (NRIgG) Chloramine T Sodium metabisulphite Sodium 125 I Sephadex G-25 Glass tubing, internal diameter 6.0 mm Ilford K5 nuclear emulsion

Although strictly the concentration of antibody used should be titrated until a plateau value of labelled cells is attained, it has been found in practice that 50 µg of pure anti-immunoglobulin

per 10 7 lymphocytes is a vast excess.

Specimen experimental protocol Anti-immunoglobulin labelling of bursa cells from six 1-day-old chicks: antiserum, rabbit anti-

chicken light chain (anti-LC) purified by acid elution from immunoadsorbent of chicken IgG; control serum, rabbit anti-KLH antibody prepared in a similar manner.

Calculation of initial concentration of reagents for protein iodination

I per 200 µg of protein in 100 µg chloramine T (final concentration). The protein concentration must be at or above 5 mg/ml to avoid excessive denaturation.

Oxidation conditions: use 37 × 10 6 Bq of 125

C H A P T E R 8: Lymphocyte structure

Specimen calculation Rabbit anti-LC and anti-KLH at 20 mg/ml initial concentration. To label 6 aliquots of bursa cells

I. To maintain 5 mg/ml protein concentration calculate permissible volume of chloramine T as follows:

at 50 µg of antibody per aliquot = 300 µg protein, use 55.5 × 10 6 Bq 125

protein used = 15 µl at 20 mg/ml, maximum permissible oxidation volume = 60 µl at 5 mg/ml.

Final oxidation mixture

15 µl protein + 15 µl 125

I + 20 µl chloramine T (total volume = 50 µl, protein concentration =

6 mg/ml).

Chloramine T used at 100 µg/ml final concentration, therefore initial concentration must be 250 µg/ml, i.e. prepare an initial solution of 25 mg chloramine T in 100 ml PBS. Reaction stopped by a two-fold excess, by weight, of sodium metabisulphite.

Final mixture

I + 20 µl chloramine T + 50 µl sodium metabisulphite. Total volume = 100 µl.

15 µl protein + 15 µl 125

Final concentration of metabisulphite = 200 µg/ml; initial concentration must be 400 µg/ml,

i.e. prepare an initial solution of 40 mg sodium metabisulphite in 100 ml PBS.

METHOD

1 Partially seal the end of two pieces of glass tubing, length 30 cm, internal diameter 0.6 cm, and plug with cotton wool.

2 Pour two columns of Sephadex G-25, height 10 cm.

3 Determine void volume and expanded sample volume of each column using 0.3 ml blue dextran (initial sample volume). Equilibrate columns with PBS.

4 Pipette out protein for iodination into pointed glass tubes.

5 Add calculated volume of sodium 125

I (carefully).

6 Add chloramine T and oxidize for 3 min at room temperature.

7 Terminate reaction by addition of sodium metabisulphite.

8 Adjust final volume to 0.3 ml with PBS.

9 Pass the iodination mixture through the Sephadex G-25 column.

10 Monitor the column effluent for the first appearance of radioactivity. ( This should be just after the void volume has left the column.)

11 Collect the labelled protein in the expanded sample volume. The radioactively labelled protein may be stored overnight at 4°C before use.

Cell labelling

1 Add 50 µg of iodinated protein (either anti-LC or anti-KLH) to aliquots of 10 7 bursal lymphocytes.

2 Incubate at 4°C for 30 min.

3 Centrifuge each aliquot of cells through a 2-ml discontinuous gradient of 50% and 100% fetal bovine serum (FBS) in tissue culture medium at 225 g for 15 min at 4°C.

8.2LYMPHOCYTE SURFACE MEMBRANE

4 Suck off the supernatant and resuspend the cells in 1 ml tissue culture medium.

5 Layer the cells onto a second gradient and centrifuge.

6 Finally resuspend the cell pellet in a few drops of FBS and prepare smears.

7 Check that the smears are adequate using a phase-contrast microscope, and adjust the cell concentration in the original suspension with FBS if required.

8 Prepare at least six smears per cell aliquot, and label the slides for identification.

9 Fix the slides, wash with running tap water for 30 min and finally air dry.

10 Dip the slides in a 1 : 5 solution v/v of Ilford K5 nuclear emulsion and dry slides in front of a fan or over silica gel overnight (in a photographic dark room).

11 Leave slides to expose in light-tight containers at 4°C. (Do not store near radioactive materials.)

Exposure time

1 Under the conditions described, one sample slide should be removed from each group after 4–5 days.

2 Develop, fix and wash the sample autoradiographs and then stain in May–Grünwald/ Giemsa.

3 Examine the slides under oil immersion. If the autoradiographs in the control groups show more than eight to 10 grains per cell, develop all the slides. If, however, the control staining is low, examine the anti-LC-treated cells. If the grain counts are clearly above the control values, develop all the slides.

4 Sample the autoradiographs at least every 4 days until satisfactory positive labelling is achieved with a low number of grains on the control cells.

5 At the end of the exposure period (usually 10–14 days under the conditions described), select at least two slides per group for grain counting. Use the following criteria: (a) the cells must be sufficiently spread so that the grains between two adjacent cells do not

overlap; (b) the cell density must be similar on anti-LC and control slides within each group; (c) the emulsion over the cells must be free from ‘fogging’ of any source.

6 Count the number of grains over at least 200 cells per group (Fig. 8.3). Record and rank the grain counts as shown in Fig. 8.4.

7 Calculate the frequency of cells within each ranked group.

Calculation of percentage of positive cells The proportion of cells showing positive labelling, i.e. grains above those expected by the non-

specific binding of labelled protein and for other non-specific reasons, may be calculated by the following equation.

For each grain-count category:

where C p is percentage of positively labelled cells, C a is proportion of cells labelled with antiserum and C c is proportion of cells labelled with control serum. Calculate the percentage positive cells in each category for each group. Plot a graph of cell fre- quency against grain counts. A specimen result and calculation is shown in Fig. 8.4.

C H A P T E R 8: Lymphocyte structure

Proportion of labelled cells Number of

% positive 0 11 17 17 10 10 8 6 2 3 4 cells

40 Anti-LC—raw data

20 Frequency

Anti-KLH—control

Anti-LC, corrected distribution—88% positive cells

Number of grains

Fig. 8.4 Histograms of grain counts from autoradiographs of 125 I anti-light chain (anti-LC) and anti- keyhole limpet haemocyanin (anti-KLH) labelling of chicken B lymphocytes.