314 ISOELECTRIC POINT OF RED BLOOD CELLS 



The test emulsion for cataphoresis was prepared by adding the 

 proper amount of acid to secure the desired H concentration to 5 cc. 

 of saccharose solution; this was then poured gently into one of the 

 second series of tubes of cells, and the mixture pipetted immediately 

 into the U-tube. In this way it was possible even at reactions near 

 the isoelectric point to observe the movement of cells before aggluti- 

 nation was complete and in many cases before macroscopic aggluti- 

 nation had appeared. For each determination 10 cc. of buffer solu- 

 tion and 10 cc. of saccharose solution, of the H concentration of the 

 tube of cells to be examined, were prepared. If the apparatus was 

 carefully manipulated the boundary of contact between the cells and 

 the fluid in the side arms remained sharp, and the distance of dis- 

 placement of the boundary during passage of current could be meas- 

 ured quite accurately with a pair of dividers. When agglutination 

 appeared, a more dilute suspension remained, the movement of which 

 could be determined likewise. 



In addition to the determinations made in the absence, as far as 

 possible, of electrolyte, measurements were made of the migration of 

 cells in the presence of sodium chloride, sodium acetate, and sodium 

 phosphate, using a dilution of 1 part of isotonic solution of these salts 

 to 4 parts of saccharose solution. The acetate and phosphate were 

 used in the form of buffer mixtures. 



Sensitized cells were prepared by adding to a 10 per cent suspen- 

 sion of cells in saccharose approximately 50 hemolytic units of a high 

 titer immune rabbit or hare serum. The reaction of the mixture was 

 about pH 6.0. After incubation for 2 hours at 37°C. the cells were 

 either used at once orwere placed in the refrigerator over night. Before 

 use the cells were sedimented twice and again made up to 10 per cent 

 concentration. The amount of electrolyte in the immune serum was 

 insufficient to cause agglutination. Kosakai^^ has shown that sensi- 

 tizer can be dissociated from red cells by extraction in salt-free solu- 

 tions of various sugars; we have been able to confirm this, but the dis- 

 sociation is not complete and the cells used in our determinations were 

 still combined with sensitizer, as shown by prompt hemolysis on the 

 addition of complement and by agglutination when added to saline 



^^ Kosakai, M., /. Immunol, 1918, iii, 109. 



