Preparation of Antibody Sensitized Sheep Erythrocytes

(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.

I. Reagent Preparation:

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).

II. Obtain Sheep Blood:

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.

III. Preparation of Sheep Red Blood Cells (RBCs):

1. Suspend the 56 mL sheep RBCs / Alsever’s suspension by gentle inversion of the bottle. Centrifuge the red blood cell suspension in sterile centrifuge tubes at 450 x g at 8 ± 3 °C for 10 min. All centrifugation steps are to be performed in a refrigerated centrifuge. 

   Original volume of sheep RBCs / Alsever’s suspension = _______ mL

2. Carefully remove the supernatant with a sterile pipette and discard.
3. First wash: Wash the sheep RBCs by gently suspending them to the original volume (56 mL) with 0.9% Sodium Chloride solution. Centrifuge the suspension at 450 x g at 8 ± 3 °C for 10 min. Carefully remove the supernatant with a sterile pipette and discard.
4. Second wash: Wash a second time with 0.9% Sodium Chloride solution and centrifuge as above. Carefully remove the supernatant and discard.
5. Third wash: Wash the cells with GVB-EDTA (G9660) by suspending them (to original 56 mL volume) and centrifuge as above. Carefully remove the supernatant and discard.
6. Add two times the original volume of GVB-EDTA (G9660) to suspend the sheep RBCs (112 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)
   
   

7. Determination of number of Red Blood Cells (RBC) / mL: A 15 fold dilution of a suspension with 10⁹ cells / mL has an absorbance of 0.7 at 541 nm.
   
   Pipette 0.5 mL from the washed sheep RBCs / GVB-EDTA (G9660) suspension using a sterile pipette into 7.0 mL of dH₂O and mix to lyse the cells. Mix and read the A₅₄₁ vs. dH₂O. If absorbance is greater than 1.0, do a 3-fold dilution by adding 1 mL of diluted cells to 2 mL of dH₂O. Account for this dilution in the formula below (Step 8) using a dilution factor of 3.
   
   

   A₅₄₁ = __________          Additional Dilution Factor (if any) ____________
   
   

8. The following calculation will determine the volume (Volume B) required to obtain an approximate cell count of 10⁹ cells / mL.
   
   

   [(A₅₄₁) / (0.7)] X [mL (Suspension A, Step 6)] X [ Additional dilution factor (Step 7)] = mL (Volume B)
   
   

9. Calculate the amount GVB-EDTA (G9660) to add to the RBCs / GVB-EDTA suspension (Suspension. A) to obtain 10⁹ cells / mL.
   
   

   [mL (Volume B, Step 8)] - [mL (Suspension A, Step 6)] = mL GVB-EDTA (G9660) to add

IV. Preparation of Rabbit Anti-Sheep Red Blood Cell Stroma (S8014) in G9660:

Note: 1 vial of S8014 is used per 100 mL of 1 x 10⁹ RBCs/mL (Volume B) in Step 8, section III. 

1. Calculate the number of vials required to treat the cells.
   

   [mL (Volume B from Step 8, Section III)] / (100 mL per vial) = vials of S8014 needed.

2. Round up to the next whole number of vials. Number of vials needed = vials
3. Using 2 mL of dH₂O, quantitatively transfer the contents of 1 vial of Rabbit Anti-Sheep Red Blood Cell Stroma (S8014) to GVB-EDTA (G9660). Continue until all vials are transferred. Then add GVB-EDTA (G9660) to bring to the same volume as the sheep RBCs / GVB-EDTA suspension (Volume B, Step 1 above). 
4. Mix for at least 5 minutes.

V. Incubation of the erythrocytes with the antibody:

1. Pour the Rabbit Anti-Sheep Red Blood Cell Stroma GVB-EDTA solution made in section IV into the suspension of RBCs from Step 9, Section III and cover. Mix with gentle swirling.
2. Incubate in a water bath at 36–38 °C with gentle shaking for 35 to 45 min.
3. Place the flask in an ice bath for 30 to 45 minutes.
4. Centrifuge the antibody sensitized sheep RBC suspension at 800 x g and 8 ± 3 °C for 10 min.
5. Carefully remove the supernatant with a sterile pipette so that no cells are lost.
6. Suspend the antibody sensitized sheep RBCs to the original volume, 56 mL, (Step 1, Section III) by adding GVB-EDTA (G9660) and transfer the suspension to sterile centrifuge tubes. Note: The material can be sealed and stored at this point at 2–8 °C overnight.
7. Centrifuge at 450 x g and 8 ± 3 °C for 10 min. Remove the supernatant with a sterile pipette.>
8. Suspend the antibody sensitized sheep RBCs to the original volume, 56 mL, (Step 1, Section III) with autoclaved 0.1 M Sucrose in GVB (G6514), (prepared in Section I).
9. Centrifuge at 450 x g at 8 ± 3 °C for 10 min. Remove the supernatant with a sterile pipette.
10. Suspend the washed antibody sensitized sheep RBCs to approximately 80 mL (1.42 times the original volume) with 0.1 M Sucrose in GVB (G6514).
11. Determine the of number of antibody sensitized sheep RBCs / mL as done in Section III, Step 7:
    
    

    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

    
    
12. Adjust the cell density to meet the experimental needs. Do this by adding more 0.1 M Sucrose in GVB (G6514) or centrifuge and remove an appropriate volume of supernatant followed by re-suspension.
13. Aliquot as needed and store the cells at 2 °C to 8 °C for up to 4 week