(Rabbit Anti-Sheep Red Blood Cell Stroma) E9383
Equipment required:
Balances accurate in the following ranges:
< 10 g: (± 1 mg)
10–200 g: (± 0.01 g)
Refrigerated Centrifuge
Autoclave
Water Bath-Shaker set at 36–38 °C
Spectrophotometer
Laminar Flow Hood
All materials should be kept on wet ice unless otherwise stated in procedure.
During the completion of this procedure, proper aseptic technique should be observed and work should take place in a laminar flow hood where appropriate. The only times that the material will be taken out of the hood is when it is capped for centrifugation and when it is covered for incubation and storage.
The process summary is as follows: Obtain fresh sheep blood and wash the cells with the reagents described. The red blood cells are adjusted to approximately 109 cells/mL. One vial of Rabbit Anti-Sheep Red Blood Cell Stroma (S8014) is used to treat each 100 mL of 109 cells/mL. The treated RBCs are adjusted to a suitable concentration and stored at 2° C to 8 °C for up to 4 weeks.
Preparation of 0.1 M Sucrose in Gelatin Veronal Buffer:
Add 5.14 g of sucrose (S0389) and 150 mL of Gelatin Veronal Buffer (G6514) into an autoclavable container. Mix until dissolved. No further adjustment is required.
Preparation of 0.9% Sodium Chloride solution:
Add 2.25 g of Sodium Chloride (S9888) and 250 mL of dH2O into an autoclavable container. Mix until dissolved. No further adjustment is required.
All buffers and containers that will come into contact with the product should be autoclaved. Autoclave the following equipment, supplies, and prepared reagents:
0.1 M sucrose in Gelatin Veronal Buffer, 0.9% Sodium Chloride solution, 2 X 250 mL glass graduated cylinders, 1 X 250 mL glass beaker, 2 x 200 mL centrifuge cups with caps, 1 x 1L Erlenmeyer flask, and 1 x 1 L glass beaker.
Note: Approximately 7 mL of whole sheep blood (14 mL of 50% Sheep blood / 50% A3551) will be used per 1 vial of S8014 (Rabbit Anti-Sheep Red Blood Cell Stroma).
Option 1: Using aseptic technique, transfer an equal volume of sheep blood directly from the animal into a sterile container of equal volume of Alsever’s Solution (A3551). For this procedure, 28 mL of sheep blood is added to 28 mL of Alsever’s solution (A3551). Volumes may be adjusted proportionally for alternate batch sizes. This will result in approximately 300–350 mL of 109 cells/mL suspension.
Option 2: Alternatively, obtain sheep red blood cells (RBCs) in any another acceptable anti-coagulant solution.
Note: Often the sheep RBCs / Alsever’s suspension is allowed to set for 4–7 days at 4 °C to ensure that any possible infection does not lyse the cells and/or only the robust cells remain. Lysed cells will be washed away in section III.
Original volume of sheep RBCs / Alsever’s suspension = _______ mL
56 mL (original volume of sheep RBCs / Alsever’s suspension) X 2 = 112 mL (Suspension. A).
[sheep RBCs / Alsever’s suspension (Step 1) mLs] X 2 = mL (Suspension A)
A541 = __________ Additional Dilution Factor (if any) ____________
[(A541) / (0.7)] X [mL (Suspension A, Step 6)] X [ Additional dilution factor (Step 7)] = mL (Volume B)
[mL (Volume B, Step 8)] - [mL (Suspension A, Step 6)] = mL GVB-EDTA (G9660) to add
Note: 1 vial of S8014 is used per 100 mL of 1 x 109 RBCs/mL (Volume B) in Step 8, section III.
[mL (Volume B from Step 8, Section III)] / (100 mL per vial) = vials of S8014 needed.
A541 = __________ Additional Dilution Factor (if any) ____________
[(A541) / (0.7)] X [ mL (Suspension A, Step 6)] X dilution factor if necessary X (1 x 109) = cells/mL
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