Características de un líder

adding 2ml of pre-warmed 37 RPMI to each petri dish, and following the addition of warm RPMI medium, the dishes were rotated gently by hand. The pooled supernate was centrifuged at 1,4 x 10® RPM for 10 minutes in a refrigerated bench centrifuge. The cell pellet of the nonadherent cells was resuspended to a concentration that was adjusted as to the original concentration of 1 x 10^ cells/ml, and 5ml of which, were dispensed into 50mm sterile petri dishes.

Preparation of adherent cells :

2ml of prewarmed 37 t Earle’s Basal salt solution (C^*, Mg

free) containing 0,25% Trypsin, (Sigma T 8003), and EDTA,

(Sigma-EDS) at a concentration of 10 moles , were dispensed into each petri dish. The dishes were rotated gently on an axial shaker at 37 C for approximately 10 minutes. Immediately after trypsinization 500 ul of calf serum were added to each petri dish to stop the action of trypsin. The contents of the petri dishes were pooled and diluted with additional amounts of medium. The contents were transferred into sterile universels and centrifuged for 10 minutes at 1.4 x 10® RPM in a refrigerated bench centrifuge. The supernate was discarded and the cell pellet was resuspended in RPMI, The cell suspension was adjusted to a concentration of 1 x 10^ cells/ml and 5ml containing the adherent cell fraction were

dispensed into sterile petri dishes , Several dishes were prepared

for the adherent and nonadherent groups. The culture dishes were transferred into incubation boxes for 16 hours at 37 °C, 5% COgin a

Leec incubator, A UIF preparation was made of each fraction as described previously. For both fractions a cell loss of 15.8% was

accounted for after centrifugation, before preparing the UIF suspension. The adherent cells were removed by mild trypsinization which was a final resort to remove them from the petri dishes. Although it would have been better to remove them by scrapping the dishes with a spatula tip that was covered with fine rubber tubing coat. The fraction of nonadherent and adherent cells of the total spleen cell (without red blood cells) was 60.91% and 23.26% repectively.

Preparation of macrophage-free spleen cell suspension (Macrophage

Free UIF);

The procedure for preparing a macrophage free cell suspension, was according to the method desbribed by Lee, (1980). The carbonyl iron powder was washed and aliquoted into bottles and sterilised on the same day of the experiment for separating cells according to their adhering ability,

lOgm of 99+% pure carbonyl iron powder <60um (Gaf.U.K.) were washed with 100ml of 0,85% saline 4 times to remove any toxic material, A U-shaped iron magnet was used to hold the iron particles down in the beaker while pouring off the saline wash. The iron powder was resuspended in 50ml physiological saline (0,2gm iron/ml saline) and was kept in suspension by constant mixing,

3ml of the iron suspension were pipetted into 100 ml sterile screw-cap bottles. The bottles and their iron contents were sterilised by autoclaving at 18lb/irf for 20 minutes. Following the autoclaving, the bottles were immediately tapped and shaken to disperse the iron; discourging it to form clumps. If clumps were to be formed, then, an alternative procedure, to avoid iron

clurapping through autoclaving, was to sterilise the iron powder by extensive washing with sterile saline containing penicillin and streptomycin, before dispensing the 3ml aliquots,

A rat spleen suspension (white cells fraction) was prepared as described previously at a concentration of 10?cells/ml. The saline contents of the bottles were aspirated by means of a sterile pasteur pipette while the iron powder was held down by a magnet. Each bottle received 8ml of the spleen suspension (8 x 10^ cells/bottle). The bottles were capped tightly and incubated in a water bath for 45 minutes at 37 °C, During the incubation period, the bottles were shaken with just enough force to suspend the iron powder completely, every 5 minutes. At the end of the incubation period, the nonadherent cell suspension was pooled from all bottles in a single sterile beaker by placing a magnet under each bottle to retain the iron particles and the adhering cells with it, A magnet was placed for a few minutes under the container, containing the pooled nonadhering cell suspension to allow residual iron powder to settle. Following the settling of the residual iron powder, the cell suspension was transferred into sterile universels and centrifuged in a refrigerated bench centrifuge at 1,4 x ICf RPM for 10 minutes. The cell pellet was resuspended in RPMI 1640 medium

without FCS to prepare a macrophage free UIF. The macrophage free

suspension was adjusted to the original concentration of 10^ cells/ml taking into account a 34.5% loss due to the centrifugation steps. 5 ml of the final preparation of the nonadherent cell suspension was dispensed into sterile 50 mm tissue culture petri dishes to produce a macrophage free UIF, The procedure for UIF preparation was described earlier.

Preparation of different molecular fractions of STD.UIF ;

Rat STD.UIF was prepared as described previously. The UIF was dispensed into an Amicon apparatus which fractionates solutions according to the molecular separating properties of the Amicon filter. Four types of Amicon Diaflo ultrfiltration membranes

(Amicon. U.K.) were used as follows;

1. A membrane that retains substances of 5 x id molecular weight and sizes above 5 x icf . Code; Amicon (XM)

2. A membrane that retains substances of 1 x itf molecular

weight and sizes above 1 x lO'^ Code; Amicon (PM)

3, A membrane that retains substances of 1 x 10^ molecular

weight and sizes above 1 x 10^ Code;Amicon (UM2)

4. A membrane that retains substances of 5 x 10^ molecular

weight and sizes above 5 x 1 0 ^ Code; Amicon (UM05)

10 ml aliquots of STD.UIF (20 ml. total volume) were dispensed into an Amicon Diaflo ultrafiltration apparatus which had a membrane of the 5 x 10"^molecular weight type. The STD.UIF was forced through the membrane under pressure [ 20/iif. 3. The

STD.UIF that passed through the filter was collected in a clean

beaker and was refractioned on a 1 x 10*molecular weight exclusion

filter. The fractioned UIF, was fractioned on a 1 x 10® molecular

weight Amicon exclusion filter. The resulting UIF was again refractioned on a 5 x 10^ molecular weight exclusion filter, and the UIF which passed through the filter was retained and coded as B500

All the respective filters (1), (2), (3) and (4) were washed in distilled water. The distilled water washings of the Amicon filters were vacuum dried in a drying machine CEdwards-Ü.K.] and each was resuspended in 20ml RPMI 1640. All the fractions of the UIF were sterilized through a 0.22um millipore filter. The fractionation of the UIF was carried out at 4 ®C.

The STD.UIF fractions are coded as follows;

5 X ICf. for molecules of 5 x lO^molecular weight and above. 10^ for molecules below 5 x 10* to ICf molecular weight, 10® for molecules below 10 to 10® molecular weight. A500 for molecules below 10® to 500 molecular weight.

B500 for molecules that passed through the 500 molecular weight exclusion filter.

All the UIF preparations were stored in sterile universels

at -20 °C for further use.

STD.UIF was heated for half an hour at 56 ®C; A sterile universal containing 20ml of STD.UIF was kept for half an hour in a water bath at 56 °C.

Preparation of dialysed Rat WCF.UIF ;

A visking dialysis tubing (size 24/32, Dept., of

Biochemistry, Univ. of St. Andrews.) which retains substances of

id* molecular weight and above was soaked in distilled water for 1 hour in a clean beaker. The dialysis tubing was transferred into a larger beaker containing double distilled water (DDW) and was heated until boiling. The dialysis tubing was boiled for 3

minutes. The dialysis tubing was later washed in DDW several times. Two knots were tied at one end of the dialysis tubing, and using a sterile 20ml syringe (Beckton Dickson. Ireland), 20ml of

Rat WCF.UIF, (STD.UIF) were dispensed into the tubing. Two knots

were tied at the top side of the tubing. The UIF was dialysed

against 200ml of RPMI 1640 containing Hepes buffer for 3 days at 4 ®C. The buffer was changed twice daily (for fresh buffer) and was

continuously stirred with a magnetic stirrer. The UIF was later

taken out of the dialysis bag and its volume recorded. The

dialysed UIF (which was of 1 x 10** molecular weight and above) was

F

sterilised through 0.22um sterile millipore filter and was stored in sterile universels at -20 ®C for further use.

Each cell culture in microtitre culture wells, which was

incubated with any type of UIF, received 0.2 uci 125IUDR, and was

replicated 5 times. The assessment of isotope uptake by cultures