Clin. exp. Immunol. (1978) 32, 361-365.

A new approach to the autoradiographic study of proliferating lymphocytes B. ZWEIMAN & R. P. LISAK Allergy and Immunology Section, Department ofMedicine and Department ofNeurology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A.

(Received 26 September 1977) SUMMARY

An adaptation of a cell filtration method for the autoradiographic study of cultured lymphocytes has been developed. The percentages of labelled cells are very similar in the filtered cell population to those obtained from replicate cultures processed by centrifugation. The filter method permitted a higher recovery of cells from small numbers in culture with better cytological detail than seen when cells were washed by repeated centrifugation, then suspended and smeared.

INTRODUCTION Autoradiographic techniques have been used extensively to investigate the uptake of isotopic precursors into suspended cells undergoing proliferation, protein synthesis, etc. This generally has been accomplished by washing the cells extensively with repeated centrifugations, to eliminate as much of the non-incorporated isotope as possible, following the pulse labelling period and before a smear of the resuspended cells is made. Reproducible readings of such cell preparations generally require the inspection of several hundred cells, especially when the percentage of labelled cells is low (Rogers, 1967). There has been increased interest recently in studying in vitro the functional activities of the relatively small number of lymphocytes in tissue compartments such as the cerebrospinal fluid (Sandberg-Wollheim, 1974; Levinson, Lisak & Zweiman, 1976). Cell losses during the repeated centrifugations used in autoradiographic techniques (noted above) frequently result in too few cells in the final smear to determine reliably the percentages of labelled cells. In addition, alterations in cell morphology induced by the repeated centrifugations and resuspensions of the cells often prevent precise descriptions of nuclear and cytoplasmic characteristics of the various cells in the preparations. Such cytological information is especially important in the investigation of fluid compartments which may contain a sizable number of non-leucocyte cells, such as the spinal fluid. We have adapted a method of cell filtration (Baringer, 1972) to the autoradiographic study of mitogen and antigen-induced lymphocyte proliferation. We feel that this method, avoiding centrifugation, leads to an improved cell yield and the preservation of morphological characteristics, as will be described in this report. MATERIALS AND METHODS Cell culture techniques. Human venous blood from eleven normal subjects was placed into heparinized bottles (final heparin concentration 10 u/ml), diluted with 2 vol. of isotonic saline and layered on a mixture of sodium diarizoate (WinthropStearns) and Ficoll (Pharmacia Labs). After centrifugation at 400 g for 40 min at room temperature, the cells at the interface ( >97% mononuclear cells) were removed, washed twice with Hanks' solution (Gibco Labs) and resuspended in culture medium. This medium consisted of Eagle's minimal essential medium, spinner culture-modified (S-MEM, Gibco Labs),

Correspondence: Dr Burton Zweiman, 512 Johnson Pavilion, University ofPennsylvania School of Medicine, Philadelphia, Pennsylvania 19174, U.S.A. 0099-9104/78/0500-0361 $02.00 (© 1978 Blackwell Scientific Publications

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with added penicillin and streptomycin (Gibco Labs) and glutamine (final concentration 2%, Gibco Labs), and 10% foetal bovine serum (Microbiologic Association). Two types of replicate cultures were set up, as described by us (Lisak & Zweiman, 1974; Levinson, Lisak & Zweiman, 1974): (a) 'macro' culture containing 2 5 x 105 mononuclear cells suspended in a final 1.0 ml volume in RTU disposable glass tubes (Becton-Dickinson Co.) with a Morton metal cap closure (Bellco); and (b) 'micro' cultures containing 5*0 x 103 cells suspended in 0-2 ml of medium in conical bottom microtitre-type culture plates (Linbro Co., catalogue No. 15-MUC-96). In each type of set-up replicate cultures contained 1/10 vol. of either: (a) PHA-P (Difco Labs) 1 Isg/ml final concentration; (b) Candida albicans (Oidomycin, Hollister-Stier Labs), undiluted; and (c) additional culture medium (control cultures). All reagents were sterile and all techniques were performed aseptically. The cultures were incubated either 3 days (for 'macro' PHA and control cultures) or 5 days (for 'macro' Candida albicans and control cultures, and for all 'micro' cultures) at 370C in 5% CO2 and 95% air. Following a 4 hr terminal pulse with 10 ili (for 'macro' cultures) or 0 25 Eli (for 'micro' cultures) of tritiated thymidine (6-7 Ci/mM, New England Nuclear Co.), the cultures were then chilled at 4VC. Cell filtration techniques for cultured cells. A vacuum manifold setup was assembled using components from the Millipore Co. (Bedford, Massachusetts). A pyrex microanalysis filter holder (catalogue No. xxl0 025 00) was fitted into a filtering sidearm 125 ml flask (catalogue No. xx 10 025 05). Gentle vacuum was provided by a Jewell vacuum pump (A. H. Thomas Co.). A three-way stopcock inserted in the tubing between the pump and the side-arm of the flask permitted rapid starting and stopping of vacuum filtration. Polycarbonate filters, 25 mm diameter, 5 zum pore size (Metricel, GA-1, Gelman Instrument Co.), were pre-incubated in 95% ethanol at room temperature for at least 5 min to enhance the swelling of the membrane and give appropriate adjustments of the pore size. Partial drying of the filter permitted the labelling of the margins of individual filters with a laundry-type marking pen. A single labelled filter was placed on the fitted glass support base, and the flared funnel was clamped in place by the aluminium clamp provided. The filters were initially washed with several passages of Hanks' solution under vacuum. An additional 5 ml of Hanks' solution was then added and the vacuum stopped. The cell culture to be filtered was resuspended and drawn into a Pasteur pipette. As the vacuum filtration was restarted, the contents of the Pasteur pipette were gradually let out into the Hanks' solution with the pipette tip directed over the centre portion of the filter. The vacuum was stopped at the point when a 2-3 ml reservoir of fluid remained on top of the filter. Two 5-10 ml vol. of additional Hanks' solution were then drawn by vacuum through the filter to wash the cells, always leaving a reservoir of at least 2 ml of fluid to avoid drying out the filter. The vacuum was stopped and 10 ml of 95% ethanol was added to the funnel. After 1 min, this ethanol was drawn through the filter until a small reservoir was left above the filter. An additional 10 ml of 95% ethanol was drawn through the filter, again leaving a 2-3 ml reservoir. The funnel was then unclamped and the filter transferred immediately blunt-tipped forceps for storage in a petri dish previously filled with 95% ethanol. Following storage at room temperature, the labelled filters were cemented to standard microscopic slides with a fastdrying glue (Permabond Pearl Co., Tokyo). In a dark-room they were then coated by dipping into NTB-2 autoradiographic emulsion (Kodak Chemicals) previously heated to 450C. The excess emulsion was drained off with the slides held vertically; the emulsion-coated slides were then dried with the help of a fan over the next 20-30 min. The slides were then stored vertically in a light-proof box at 4VC for 1 week. They were then developed and fixed in the dark with D-10 Kodak developer (6 min) and Kodak rapid fixer (6 min), were washed repeatedly in distilled water and then stained with a modified Papanicolaou technique. Following the clearing of the filters in zylene for 10 min, the filters were immersed in Eukitt mounting medium (Calibrated Instruments, Ardsley, New York) for 20 min and transferred to alcohol-cleaned slides. A cover slip was applied immediately with gentle pressure to squeeze out excess mounting medium, which was then removed. After storage overnight, the edges of the cover slip were coated with a clear nail polish to make an air-tight seal. Smear technique for cultured cells. Culture plates or vials were centrifuged at 4VC at 200 g for 10 min. The supernatants were removed carefully and the cell buttons washed twice by resuspension in Hanks' solution (containing 5% foetal calf serum) and repeated centrifugation. The cell buttons were then resuspended in foetal calf serum (2 drops for 'macro', 1 drop for 'micro' cultures) and then transferred to appropriately labelled alcohol-cleaned slides with Pasteur pipettes. The drops were smeared with the drawn-out tip of the pipette over a limited area of the slide, which was then allowed to dry at room temperature. The smear area was permanently identified by scoring the reverse side of the slide with a glass-marking pen. Once dried the slides were stained with a 5% methylene blue solution for 30 sec, were washed off immediately and mounted in Permount (Fisher Scientific Co.). All slides were code-labelled. 200 were counted and the determination made of the percentage of mononuclear cells overlaid with at least three times the number of silver grains seen in an average background area of the same diameter. After the codes were broken, the percentages of labelled cultures in replicate-filtered cell cultures were compared with companion cultures in which smear preparations were used for autoradiography. Correlations were then attempted by linear regression analysis.

RESULTS The percentage of labelled cells in PHA-induced 'macro' cultures subsequently processed by vacuum filtration and smear techniques are shown in Table 1. The percentages labelled in replicate cultures processed by the two techniques were very similar (correlation coefficient r = 094, P< 0.001). Compari-

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son of the labelling percentages in the filter and smear-processed replicate cultures were also quite similar for antigen-stimulated (r = 0-92, P< 0.01) and control (r = 0 70, P< 0-01) cultures (Table 1). Although the percentages of labelled lymphocytes were quite similar using the two processing methods, there were distinct differences in the qualitative appearance of the cells themselves. Cells resuspended and then smeared were more frequently clumped than the filtered cells, with a rounding of individual cells as compared with those filtered. This was particularly true in the case of PHA-incubated cultures. In addition, the staining characteristics of the processed cells were quite different. The nuclearcytoplasmic distinctions and cell borders were much more defined in the filtered cells, the nucleoli often visible. The distinction in size among individual stimulated and non-stimulated cells were readily appreciated and easily measured. By comparison, the cytoplasm in the smeared cells was frequently less well-defined or even barely visible, with less distinct cell borders and nuclear-cytoplasmic relationships. TABLE 1. Comparison of the percentages of labelled cells-filtered and smeared lymphocyte cultures

Subject (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (1 1)

PHA F* St

26t 28 42 45 36 46 63 58 55 60 43

27 26 43 46 32 50 62 58 68 56 44

Antigen F S -

-

-

-

-

-

-

-

-

-

-

-

4 7 15 6 9

5 6 20 8 7

Control F S 1 0 0 1 0 3 1 0 1 0 1

0 0 1 1 0 1 0 1 1 0 0

* Cultured cells aspirated onto filters with subsequent autoradiography. t Cultured washed and smeared with subsequent autoradiography. t Percentage oflabelled cells.

Another difference was seen in the comparison of filter and smear processing of those small numbers ofcells obtained from 'micro' cultures of 5 x 103 mononuclear leucocytes. The filtered cells were present in sufficient numbers to readily determine the percentage labelled. Comparison of 'macro' and 'micro' PHA-incubated cultures of the cell suspensions, which were subsequently filter-processed, showed similar percentages of labelled cells (Table 2). By contrast, it was quite difficult to identify as many as fifty well-defined cells in the thick smears of centrifuge-processed 'micro' cultures, preventing a reliable comparison of labelling with the adequate cell numbers obtained from the 'macro' cultures by centrifugation. DISCUSSION This report describes a useful adaptation of cell cytology techniques to the autoradiographic investigation of cultured lymphoid cells. Use of this technique permits a higher recovery and therefore a more reliable analysis of the isotopic labelling of the small numbers of cells that would be available from cerebrospinal fluid, small tissue specimens obtained with fiber-optic instruments, etc. Our comparative analyses of filtered and centrifuged cells from replicate cultures showed that there was very likely no

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B. Zweiman & R. P. Lisak TABLE 2. Comparison oflabelled cells in 'macro' and 'micro' cultures-filter-processed

Subject

'Macro'* 'Micro't

(1) PHA (1) Control (2) PHA (2) Control (3) PHA (3) Control (4) Antigen (4) Control (11)PHA ( 1) Control (12) PHA (12) Control (13) PHA (13) Control (14) Antigen (14) Control

26: 1 28 2 42 0 4 0 43 1 24 0 24 1 4 0

22 2 29 1 41 0 6 0 42 1 22 0 28 1 6 0

2 x 105 cells per ml. t 1 x 104 cells/01 ml. t Percentage of labelled cells.

*

selective loss of small lymphocytes during the filtration process. In addition, washing the cells without repeated centrifugations appears to lead to flatter, better preserved cells. The superior cytological appearance of the filtered cells does not appear to be a simple function of the Papanicolaou staining method used. Use of this and other staining methods on the smeared cells did not result in noticeably better appearance of the centrifuged cells compared to the methylene blue method reported. Also, prior fixation of the smears in glutaraldehyde before methylene blue staining did not enhance the appearance of centrifuged cells to near the level of definition seen in well stained filtered cells. Such cellular distinctions are of particular importance in helping to distinguish lymphoid and non-lymphoid cells in a heterogeneous population from a tissue or body fluid compartment. Attempts to date to obtain, by other methods, adequate numbers of well-defined cells from the small populations cultured have led to less satisfactory results. For example, a reasonable number of cells were deposited on microscope slides inserted into a cyto-centrifuge (Stokes et al., 1975). However, the repeated centrifugation required to wash non-incorporated isotope from the field led to a considerable flattening and distortion of some cells, even when relatively low centrifugation speeds were used. Although the filtration method proved superior for the purposes noted here, several limitations and considerations should be mentioned. Excess drying of the filter does often lead to some cellular distortion. For this reason, the filters were dipped in autoratiographic emulsion while still moist. However, the overlying emulsion did not prevent some drying of the filter during the drying period of the developing process. Numerous attempts at interposing a barrier such as rigid cover slip or sprayed cover slip material have resulted in unacceptable interference with the tritium activation of the overlying emulsion. Studies utilizing cultures with thymidine labelled with carbon-14 instead of tritium are now underway to take advantage of the higher energy emission of carbon-14, that may not be blocked by the interposed barrier. Another factor to consider is the staining after the emulsion has been applied and developed. There is some staining of the emulsion itself, which is variable in degree and can be reduced by some experimentation with the duration of incubation with the individual stain components in the Papanicalaou method. Also, care must be taken during the staining itself to avoid a partial shedding of the emulsion due to

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overly vigorous agitation of the slides. The Papanicalaou method does not lend itself to staining prior to application of the emulsion, since the developing process leads to decolouration. Other pre-emulsion staining methods are being explored. However, for the several reasons mentioned, we feel that the technique described represents a definite advance in the autoradiographic approaches to the culture of small numbers of suspended cells. Hopefully, future modifications will yield an even further improvement. The authors would like to thank Ms C. Cohen and Ms K. Manthorne for their technical assistance. Supported by NIH grants Nos PO NS 11037, and NS 075-9, NIH Contract No. NO1-HR42950 and the MS Society grant 894-B-2.

REFERENCES BARINGER, J.R. (1972) A simplified method for spinal fluid of experimental allergic encephalomyelitis. Cell. Immunol. cytology. Archs Neurol. 22, 305. 14,242. LEVINSON, A.I., LisAK, R.P. & ZwEimAN, B. (1974) A ROGERS, A.W. (1967) Techniques of Autoradiography, p. 68. Elsevier Publishing Co., Amsterdam. microtechnique for PHA transformation of 5,000 separated lymphocytes. Cell. Immunol. 14, 321. SANBERECWOLUiMIM, G. (1974) Immunoglobulin synLEVINSON, A.I., LIsAK, R.P. & ZwEIMAN, B. (1976) Immuthesis in vitro by cerebrospinal fluid cells in patients with multiple sclerosis. Scand.J. Immunol. 3, 717. nologic characterization of cerebrospinal fluid lymphoSTOKES, H.B., O'HARA, C.M., BUCHANAN, R.D. & OLSON cytes: preliminary report. Neurology, 26, 693. W.H. (1975) An improved method for examination of LIsAm, R.P. & ZwEIMAN, B. (1974) Immune responses to myelin basic protein in mycobacterial-induced suppression cerebrospinal fluid cells. Neurology, 25, 901.

A new approach to the autoradiographic study of proliferating lymphocytes.

Clin. exp. Immunol. (1978) 32, 361-365. A new approach to the autoradiographic study of proliferating lymphocytes B. ZWEIMAN & R. P. LISAK Allergy an...
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