Appendix 1:  Preparation of Washed Dogfish Erythrocytes; Removal of Leucocytes.

(A) APPENDIX 1 PROCEDURE: ERYTHROCYTE WASHES

(using 80 ml blood, obtained from 2 smooth dogfish of moderate size; ~36-39 inches [see Appendix 3])

NOTE: For solutions needed, see section B below; WS = Working Solution; WS#1, Elasmobranch Ringers Solution; WS#2, = Heparinized Elasmobranch Ringers Solution; WS#3 = Dilute Heparinized Elasmobranch Ringers; WS#4 = Sucrose-Ringers solution

NOTE: Pre-warm all solutions to ~20-23°C before use.

Centrifuges: We use the International Equipment Corporation (IEC) Model HNS-II (with #957 4-place rotor that takes 100 ml calibrated conical glass tubes) and Model CS (with #277 4-place swinging bucket rotor that holds 200 ml bottles).

  1. Add 80 ml blood to 80 ml Heparinized Ringers (WS#2; 100U/ml); total vol. = 160 ml.
  2. Centrifuge 4 x 40 ml in 100 ml glass conical centrifuge bottles, HNS-II centrifuge (or equivalent), 2 minutes at top speed (~750 g).
  3. Swirl up upper white layer and siphon off white cells.
  4. Resuspend up to 40 ml per bottle with dilute Heparinized Ringers (WS#3) and pool to 160 ml.
  5. Set up four 200 ml conical plastic centrifuge bottles each containing 150 ml of Sucrose-Ringers (WS#4; it is convenient to prepare these earlier, and set them aside for later use).
  6. Layer 4 x 40 ml onto Sucrose Ringers (this can be done by hand pipetting, but is much easier using funnels modified so that their delivery tubes curve slightly upward at the tips, producing a gentle layering flow).
  7. Centrifuge 1 minute, CS centrifuge (or equivalent), top speed (~2400 RPM, ~1300 g).
  8. Aspirate off supernate (including remaining suspended erythrocytes) and resuspend with Elasmobranch Ringer's, to 150 ml per bottle.
  9. Centrifuge for 2 minutes as previously.
  10. Remove ~half of the supernate volume, swirl up white cells (if any); aspirate off and discard the remaining supernate containing white cells.
  11. Resuspend and pool to 600 ml with Elasmobranch Ringers (150 ml/tube).
  12. Divide equally into four tubes and centrifuge for 2 minutes as before.
  13. For working erythrocyte suspension, resuspend and pool in 600 ml total volume with Ringer's (WS#1); for preparation of cytoskeletons, do not resuspend in Ringer's, but proceed directly to steps in Appendix 2.

(B) APPENDIX 1 RECIPES: WORKING SOL'NS =WS; STOCK SOLN'S=SS

WS#1, Elasmobranch Ringers Solution (Cavanaugh, 1975); TOTAL VOL. TO BE PREPARED =1500 ml

STOCK SOLUTIONS NEEDED

STOCK SOL'N INGREDIENT(S) AMOUNT/VOL FINAL STOCK CONC.
SS#1 NaHCO3 14.25 g/250 ml 0.68M
SS#2 NaH2PO4.H2O 7 g/100 ml 7%
SS#3 CaCl2.2H2O 14.7 g/100 ml 1M

NOTES
(1) Storage conditions - refrigerate SS#1,2,3

For WS#1, Elasmobranch Ringers Solution

INGREDIENT AMOUNT/VOL FINAL CONC.
NaCl 24.57 g 0.28M
Urea 32.4 g 0.54M
KCl 1.34 g 0.01M
NaHCO3 10 ml SS#1 0.0045M
NaH2PO4.H2O 1.5 ml SS#2 0.007%
CaCl2.2H2O 15 ml SS#3 0.01M
*dextrose 1.5 g -

*do not add until just before use

NOTES

  1. Mix first three ingredients together with water to 500 ml; add next two together in another 500 ml; add CaCl2 last, diluted in final 500ml. This avoids precipitation of calcium salts.
  2. Urea, not usually present in physiological solutions, is a normal major constituent of elasmobranch blood.
  3. Accuracy of graduated cylinder is sufficient
  4. Storage conditions - Refrigerate WITHOUT ADDING THE DEXTROSE. Solution will be usable for about a week.
  5. Add 1.5 g dextrose (mix thoroughly) before use. With dextrose present, bacteria will grow more rapidly in the solution.

WS#2, Heparinized Elasmobranch Ringers Solution, 100U/ml; TOTAL VOL. TO BE PREPARED = 160 ml

(Ref. for WS#2,WS#3,WS#4: Cohen and Ginsburg,1986).

STOCK SOLUTION NEEDED

STOCK SOL'N INGREDIENT AMOUNT/VOL FINAL STOCK CONC.
SS#4 heparin 100,000U/50ml 2000 U/ml

NOTES
1. Storage conditions - FREEZE in conveniently sized aliquots (e.g., 8 ml) 2. We use Sigma #H3125 (heparin, sodium salt, from porcine intestinal mucosa); Dilute 100,000 Units (bottle contents) with 50 ml H2O

For WS#2, Heparinized Elasmobranch Ringers Solution, 100 U/ml

INGREDIENT(S) AMOUNT/VOL FINAL CONC.
heparin stock, SS#4 8 ml 100 U/ml
Elasmobranch Ringer's WS#1 152 ml -

NOTES
(1) Bring 8 ml Heparin stock up to 160 ml with Ringers; divide equally into two graduated cylinders. Blood will be mixed directly with one half; the other half will be used to make dilute Heparinized Elasmobranch Ringer's (WS#3).

WS#3, Dilute Heparinized Elasmobranch Ringers (50U/ml); TOTAL VOL. TO BE PREPARED = 160ml
INGREDIENT(S) AMOUNT/VOL FINAL CONC.
Heparinized Ringer's WS#2 80 ml 50 U/ml
Elasmobranch Ringer's WS#1 80 ml -

WS#4, Sucrose-Ringers; TOTAL VOL. TO BE PREPARED = 600ml

STOCK SOLUTION NEEDED

STOCK SOL'N INGREDIENT(S) AMOUNT/VOL FINAL CONC.
SS#5 Sucrose 342.3 g/L 1 M

NOTES
(1) Storage conditions - refrigerate.

For WS#4, Sucrose-Ringer's, 600 ml

INGREDIENT(S) AMOUNT/VOL FINAL CONC.
Sucrose stock (SS#5) 300ml 0.5 M
Elasmobranch Ringer's (WS#1) 150ml Ringer's/4
H20 150ml -