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2. Dilutions

3. Handling of Animals, Immunizations

4. Antibody-Antigen Reactions

5. Quantitative Precipitin Test

8. Dot Blot

9. Complement

10. Blood Cell Counts

11. Differential Blood Cell Counts

12. Trypan Blue Exclusion and Cell Cytotoxicity

13. Isolation of Whole Mononuclear Cells From Blood

14. Rosettes With Sheep Red Blood Cells

1. Label 7 microfuge tubes by numbering them from 1 to 7. Add 700 µl of 0.85% NaCl to tubes 2 through 7.
2. Add 700 µl of 0.1% saponin to tubes 1 and 2.
3. Mix the contents of tube 2 thoroughly and transfer 700 µl to tube 3. Mix and transfer 700 µl to tube 4, and so on to tube 6. Discard 700 µl from tube 6.
4. Add 700 µl of 2% erythrocytes to each tube.
5. Mix and incubate the tubes in a 37^o C water bath for 30 min, then centrifuge at 1500 X g for 3 min.
6. Record hemolysis on the basis of +, 2+, 3+, and 4+.
7. The titer is the highest dilution of saponin that gives any evidence of the desired reaction.
8. Your TA will demonstrate the change in color in this dilution scheme using a spectrophotometer and provide you with data that you will plot.
9. Report your findings:

1. Label 5 microfuge tubes by numbering them from 1 to 5. Add 900 µl of water to each of the tubes.
2. Add 100 µl of the methylene blue dye solution to tube 1 (10^-1).
3. Mix the contents thoroughly and transfer 100 µl from tube 1 to tube 2.
4. Mix and transfer 100 µl to tube 3, and so on to tube 5. Discard 100 µl from tube 5 to maintain a constant volume in all tubes.
5. Visually compare all 5 tubes. The color intensity should diminish as the methylene blue is sequentially diluted.
6. Your TA will demonstrate the change in color in this dilution scheme using a spectrophotometer and provide you with data that you will plot.
7. Report your findings:

See Dilutions

1. Rabbits
2. Mice
3. Video on animal use and care
4. NIH Site for Animal Care and Use
5. Institutional Animal Care and Use Committee Click on Research, then on Animal Care and Use at the site.
6. Quiz on Animal Care and Use
7. The UTEP "Animal Care and Use" Form
8. Vaccines
9. Adjuvants
10. Demonstration of animal handling and injection routes.

1. Anti-BSA (1.2 ml of 1:2 dilution per pair)
2. 0.01% BSA (6 ml per pair)
3. Saline (20 ml per pair)
4. Pasteur pipets (2 per pair)
5. Durham tubes (5 per pair)
6. Microfuge tubes (10 per pair)
7. Microfuge
8. 37^o C water bath
9. Micropipets, tips
10. Microfuge tube racks, incubation racks, Durham tube holders

1. Label 5 microfuge tubes.
2. Add 100 µl of Anti-BSA to each tube.
3. Add 6 µl of 0.01% BSA to tube 1, 12 µl to tube 2, 25 µl to tube 3, 50 µl to tube 4, and 100 µl to tube 5.
4. Add sufficient saline to each tube to give a final volume of 200 µl.
5. Mix thoroughly and incubate for 15 min in a 37^o C water bath.
6. Centrifuge for 1 min in the microfuge.
7. Set up 5 Durham tubes in a rack.
8. Add approximately 60 µl of anti-BSA to each Durham tube.
9. Carefully overlay the anti-BSA with 60 µl of supernatant from the tubes in step 6, supernatant 1 to tube 1, supernantant 2 to tube 2, and so forth.
10. Look for formation of a ring of precipitate at the interface that occurs within 15 min.
11. Choose the tube that has a ring of precipitation. This represents antigen excess.

1. Set up four microfuge tubes.
2. Add two times the amount of antigen determined above to give maximum precipitation (one tube below that which gave the interfacial ring precipitin)to tube one. For example if the tube with 50 µl of antigen gave an antigen excess reaction, then the tube with 25 µl gave maximum precipitation and 50 µl should be pipetted to tube one. 
3. Add the exact amount of antigen determined above to give maximum precipitation to tube two.
4. Add half this amount of antigen to tube three.
5. Add half the amount of antigen in step 4 to tube four.
6. Add 100 µl of anti-BSA to each tube.
7. Add enough saline to make a total volume of 200 µl in each tube.  Mix.
8. Incubate in a 37^o C water bath for 1 hour.
9. Incubate in the cold until next week. 

1. BSA (100 µg/ml; 1 ml per pair)
2. Microfuge tubes (11 per pair
3. Micropipets, tips
4. Microcentrifuge
5. Spectrophotometer
6. Bradford Reagent (2.5 ml per pair)
7. 0.85% NaCl (10 ml per pair)
8. Cuvettes (11 per pair)
9. Vortex mixer

1. Prepare a standard curve with BSA by transferring 10 µl, 20 µl, 50 µl, 100 µl, 150 µl, and 200 µl of BSA to six microfuge tubes. Add the required amount of saline to bring the volume in each tube to 800 µl. Add 800 µl of saline to a seventh tube to serve as a blank. What is the concentration of BSA in each sample?
2. Add 200 µl of dye reagent concentrate to each tube and vortex.
3. Allow the tubes to incubate for at least 5 min, but not longer than 1 hour at room temperature.
4. Transfer the contents of the blank to a cuvette and zero the spectrophotometer which has been set at a wavelength of 595 nm.
5. Transfer the contents of the remaining tubes to clean cuvettes and take a readings of each.
6. Plot the absorbance values of each sample versus the protein concentrations of each tube for the standard curve. Use the Macintosh computer in Rm 411 for plotting your figure.

1. Centrifuge the tubes of anti-BSA BSA precipitations prepared last week for 2 min in the microcentrifuge.
2. Carefully remove the supernatant from each tube with a micropipet using a tip with a long, narrow stem. It is critical that no part of the precipitate from any tube be lost. Discard the supernatants. (Normally, the supernatants would be tested for antibody or antigen). 
3. Drain all excess water from the precipitate. Be careful not to remove any of the precipitate.  It is critical that no part of the precipitate from any tube be lost.
4. Dissolve the precipitates in the four tubes in 1.0 ml of saline, then transfer 50 µl, to four microfuge tubes.
5. Bring the solution up to 800 µl with saline
6. Add 200 µl of dye reagent concentrate to each tube and vortex.
7. Allow the tubes to incubate for at least 5 min, but not longer than 1 hour at room temperature.
8. Transfer the contents of the four tubes to four cuvettes and read the samples with the spectrophotmeter.
9. Determine the concentration of the sediments using the standard curve plotted above. Remember to convert to total µg in the original precipitate samples.
10. Draw a precipitation curve, plotting total protein vs. BSA added to each tube (last week's exercise).  Determine the zone of antigen and antibody excess.  Determine the equivalence zone.
11. Turn in your work next week.

1. 2% agar in barbital buffer, 12 ml/pair
2. Anti-BSA, 120 µl/pair
3. BSA (200 µg/ml; 200 µl/pair); unknown samples 20 µl/pair
4. PBS, 10 ml/pair
5. Thin plates
6. Double diffusion plates
7. Suction flask with gel punch
8. Humidified chamber
9. Micropipets, tips, microfuge tube racks
10. 56^o C water bath.
11. Microfuge tubes, 5/pair

1. Melt agar in a boiling water bath and transfer it to a 56^o C water bath.
2. Dilute the antiserum in PBS (100 µl of antiserum to 2.4 ml of PBS)and warm to 56^o C.
3. Add the diluted antiserum to 1.5 ml of agar at 56^o C and mix well.
4. Add the agar onto a thin plate on a level surface and allow to set.
5. Punch 6 wells in the set agar. The wells should be 2 mm to 3 mm in diameter, and must have absolutely vertical sides.
6. Remove the agar plug with suction.
7. Make convenient solutions for the standard containing 50, 100, 150, and 200 µg BSA per ml. Hint: 40 µl BSA + 0 µl PBS; 30 µl BSA + 10 µl PBS; 20 µl BSA + 20 µl PBS; 10 µl BSA + 30 µl PBS.
8. Fill each of four wells with standard solutions of 50, 100, 150, and 200 µg/ml of BSA. Add the two unknowns to the two other wells.  Fill the wells quickly until the meniscus just disappears.
9. Leave the plate in a humidified chamber at room temperature until Monday.
10. On Monday, measure the diameter of each ring and plot the diameter vs. the concentration of the known samples for the standard curve.  Measure the diameter of the unknown and determine their concentrations.
11. Report your results.

1. Melt the agar in a boiling water bath.
2. Pour the agar into a plate and allow the agar to set.
3. Punch the pattern of the template on three different sites on the agar using the gel punch and suction.
4. Fill the center well of one of the patterns with anti-BSA. Add 7 µl of the standards into four of the wells in the periphery of the pattern.
5. Fill the center well of second of the patterns with anti-Crotalus venom antiserum. Add 7 µl of different rattlesnake venoms into the six of the wells in the periphery of the pattern. Make sure to note which venoms were added to which well.
6. Leave the plate in a humidified chamber at room temperature until Monday.
7. Report your results by drawing the precipitin lines that developed.

1. Barbital buffer, pH 8.6
2. Cellulose acetate electrophoresis strips
3. Ponceau S protein staining solution
4. Wash solution, 5% acetic acid
5. Power supplies, electrophoresis setup
6. Staining troughs, wash troughs
7. Serum sample
8. Micropipets, tips,
9. Microfuge tubes
10. Spectrophotometer, cuvettes
11. Scissors
12. 13 x 100 mm test tubes
13. Clearing solvent (1 part formic acid to 9 parts dimethylsulfoxide)
14. Forceps

1. Set up the electrophoresis unit as described by your laboratory assistant.
2. Fill the buffer chambers with electrophoresis buffer.
3. Place a strip of cellulose acetate in the buffer in one chamber and allow it to saturate with buffer. Use forceps to handle the strip.
4. Remove the strip and blot off the excess buffer.  Immediately apply 2 µl of the serum sample to the cellulose acetate strip.  Apply the sample about 1.5 cm from the negative pole and in a narrow line parallel to the end. Specimen application is critical and improper application will result in poor results.
5. Place the strip across the buffer unit with the ends in the buffer.
6. Apply  power of 10 to 30 mA at 200 to 300 volts.  Run for 60 minutes.
7. Remove the strip after electrophoresis and stain the proteins on the strip with Ponceau S solution for 5 min.
8. Wash the strip in 5% acetic acid until all of the background stain is removed.
9. Make a drawing of your results.
10. Dry the acetate strip at 37^o C.
11. Estimate the amount of protein in each protein band by cutting the strip into sections, then placing each section into separate 13 X 100 mm test tubes.
12. Add exactly 5 ml of clearing solvent to each tube.  The solvent will dissolve the cellulose acetate.
13. Determine the absorbance of each solution with the spectrophotometer at 520 nm.  Set the absorbance at 0 with a blank containing the clearing solvent.
14. The absorbance of each sample should be proportional to the amount of protein in the fraction.
15. Plot absorbance vs. fraction labeling the fraction accordingly.    

Materials:

1. Crotalus venoms: 1 mg/ml of C. m. molossus (northern blacktailed rattlesnake) venom; 1 mg/ml of C. atrox (western diamondback rattlesnake) venom.
2. Rabbit anti-Crotalus venom antibodies: anti-M4 and anti-M5 recognize metalloproteinases in C. m. molossus venom.
3. Goat anti-rabbit IgG conjugated with horse radish peroxidase
4. Immobilon membranes
5. Blot developing tray
6. 0.85% NaCl
7. Developing solution  (H₂O, 8 ml; Buffer 0.9 ml; H₂O₂, 0.1 ml;   4-Chloro1-Naphthol, 1 ml).
8. H₂O₂
9. Blotto (Powder milk, 25 g; PBS (7.2), 473 ml; Thimerasol, 5 ml; AntiFoam A, 50 µl).
10. 70% methanol
11. Scapel

Method:

1. Cut a strip of Immobilon membrane with the scapel so that it fits in one of the troughs in the blot developing tray. Do not touch the membrane with your hands.
2. Wet the membrane in alcohol for approximately 30 seconds, then transfer it to water.
3. Remove the membrane from the water and place it on a paper towel and blot but do not let it dry.
4. Place a 1µl drop of each of the venoms provided on the membrane.   Make sure that you know which drop corresponds to each venom.
5. Place a 1µl drop of saline on the membrane as a control.
6. Place the membrane in one of the troughs in the blot developing tray.
7. Add 1 ml of Blotto to the trough to cover the membrane, and incubate for 15 min.
8. Add 20 µl of rabbit anti-Crotalus venom antibodies directly to the Blotto. Incubate for one hour.
9. Remove the Blotto by suction.  Wash the membrane repeatedly with water to remove unreacted anti-Crotalus antibodies.
10. Add 1 ml of Blotto to the trough to cover the membrane, then add 20 µl of goat anti-rabbit IgG conjugated to horse radish peroxidase to the Blotto.  Incubate for 1 hour.
11. Prepare the developing solution 10 min prior to step 12 below by mixing the developing components listed under "Materials".
12. Remove the Blotto by suction.  Wash the membrane repeatedly with water to remove unreacted anti-rabbit IgG antibodies.
13. Add developing solution to the trough and incubate until the dots develop.
14. Report your results.  Why is the membrane treated with methanol at the start of this exercise? Why is the membrane treated with Blotto?  Did the two antisera recognize all the venoms?  What can you say about this?

Preparation of Sheep Red Blood Cells (E)

1. Place 2 to 3 ml of gently resuspended cells in a 15 ml conical centrifuge tube. Add 5 to 6 ml of 0.85% saline and centrifuge at 400 RPM for 10 to 15 min. Aspirate the supernantant and wash again with 5 to 6 ml of 0.85% NaCl.
2. Wash as above with 5 to 6 ml of EDTA-GVB.
3. Wash as above with 5 to 6 ml GVB++. Aspirate the supernatant and add a known volume of GVB++ to the packed cells. Gently resuspend the cells with a Pasteur pipet. Keep the suspension in an ice bath.
4. Mix 50 µl of cell suspension with 500 µl of distilled water. Add 1 ml EDTA-GVB, mix and read with a spectrophotometer at a wavelength of 541 nm. An OD reading between 0.68 to 0.72 is approximately equal to 1 X 10⁹ cells. Make appropriate adjustment to the cell concentration by adding additional GVB++, or centrifuging and removing supernatant until the OD = 0.68 to 0.72 after lysing a 50 ml cell aliquote with 500 ml of distilled water. Use GVB++ to baseline the spectrophotometer.

Volume of GVB++ can also be computed mathematically by means of the following formula, where

V1 = the original volume in which the cells were suspended

V2 = the volume of cell suspension that should contain 1 X 10⁹ cells/ml

V3 = the volume of GVB++ to be added to the suspension in order to obtain an OD between 0.68 and 0.72 at 541 nm.

(OD of V1/0.703)(V1) = V2

V2 - V1 = V3

Check the OD of a lysed 0.1 ml aliquot after addition of GVB++ to the original cell suspension.

1. Prepare a 1:1000 dilution of anti-SRBC antibodies in GVB++ (= hemolysin).
2. Mix the 1 X 10⁹ suspension of SRBCs with an equal volume of hemolysin.
3. Incubate at 37^o C for 30 min with periodic mixing.
4. Transfer to an ice bath and incubate for 30 min with periodic mixing.
5. Centrifuge at 700 X g for 10 min and aspirate the supernatant.
6. Wash with GVB++. Centrifuge and aspirate the supernatant.
7. Resuspend the cells in a known volume of GVB++ and follow the procedure in Step 4 of the "Preparation" to adjust the cell number to 1 X 10⁹. Keep the suspension in an ice bath.

                  (OD complete lysis - OD cell control)

1. Add an equal volume of venom (at 1 mg/ml) to undiluted complement and incubate at 37^o C for 30 min.
2. Add an equal volume of GVB++ to undiluted complement and incubate as above.
3. Dilute the venom-treated and untreated complement with GVB++ to give a final 1:5 dilution of complement.For example: 80 ml of 1 mg/ml venom + 80 ml Of undil complment.After incubation add 240 ml of GVB++ (80 ml of complement in a total volume of 400 ml = 1:5 dilution).
4. Set up the assay as in "Titration of Complement", using the treated  complement. Use the untreated complement (from step 2 above) as a    control to determine the CH₅₀.

 Materials:

1. Unopette Microcollection System
2. Blood collected with an anticoagulant
3. Neubauer Hemocytometers
4. Microscopes
5. Kim Wipes

Method:

Erythrocyte Counting

1. Puncture the Diaphragm - Using the protective shield on the capillary pipette, puncture the diaphragm of the reservoir as follows: a) Place the reservoir on a flat surface.  Grasping the reservoir in one hand, take the pipette assembly in the other and push the tip of the pipette shield firmly through the diaphragm in the neck of the reservoir, then remove. b) Remove the shield from the pipette assembly with a twist.
2. Add the Sample - Fill the capillary with whole blood and transfer to the reservoir as follows: a) Holding the pipette almost horizontally, touch the tip of the pipette to the blood.  The pipette will fill by capillary action.  Filling is complete and will stop automatically when the blood reaches the end of the capillary bore in the neck of the pipette. b) Wipe the excess blood from the outside of the capillary pipette, making certain that no sample is removed from the capillary bore.  c) Squeeze the reservoir slightly to force out some air.  Do not expel any liquid.  Maintain pressure on the reservoir.  d) Cover the opening of the overflow chamber of the pipette with the index finger and seat the pipette securely in the reservoir neck.  e) Release the pressure on the reservoir.  Then remove the finger from the pipette.  Negative pressure will draw blood into the diluent.  f) Squeeze the reservoir gently two or three times to rinse the capillary bore, forcing the diluent up into, but not out of, the overflow chamber, releasing pressure each time to return the mixture to the reservoir. g) Gently invert a few times to thoroughly mix the blood with the diluent.
3. Charge the Hemocytometer - If the sample has been allowed to stand, mix the diluted blood thoroughly to resuspend the cells.  a) Convert to a dropper assembly by withdrawing the pipette from the reservoir and reseat securely in the reverse position.  b) To clean the capillary bore, invert the reservoir, gently squeeze the sides and discard the first three or four drops.  c)  Carefully charge the hemocytometer with diluted blood by gently squeezing the sides of the reservoir to expel the contents until the chamber is properly filled.
4. Count and Calculate - An erythrocyte count is done with a Neubauer hemocytometer as follows: a) Using 430X magnification, count the erythrocytes in the four corner squares and the one center square within the large center square of the chamber.  b)  Multiply the total number of cells counted in the five squares by 10,000.  EXAMPLE:  If 250 cells are counted, the total count is: 250 X 10,000 = 2,500,000 erythrocytes/cu mm.
5. Limitation of the Procedure - A highly elevated erythrocyte count may make accurate counting difficult.  In this instance, a secondary dilution should be made.  When calculating the total count, adjust the formula to allow for the secondary dilution.
6. Technical Note - Cells and diluent(s) must be adequately mixed and counting chambers should be properly filled if errors in manual counting procedures are to be avoided.

White Blood Cell Counting

1. Follow the procedure as above for erythrocyte counting, but use the Unopette for white blood cells.
2. Count and Calculate - A white blood cell count is done with a Neubauer hemocytometer as follows: a) Using 430X magnification, count the white blood cells in the four large corner squares, each of which contains sixteen smaller squares. b)  Divide the count by 4 and multiply the total number of cells counted by 20.  EXAMPLE:  If 65 cells are counted, the total count is: 440 X 20 = 8,800 white blood cells/cu mm.

Exercise 11. Differential Blood Cell Counts

Materials:

1. SureStain Wright (Fisher Diagnostics CS432)
2. Distilled water
3. Microscope Slides

Method:

1. Prepare a thin blood smear and allow to air dry.
2. Dip the slide in SureStain Wright for 5 to 15 seconds.
3. Dip the slide in distilled water for 10 to 20 seconds.
4. Rinse the slide by dipping in fresh distilled water for a few seconds and air dry.
5. Observe the cells by using oil immersion. 
6. Do a differential cell count by counting 100 cells and keeping track of the number of neutrophils, basophils, eosinophils, monocytes, and lymphocytes.

See differential staining

Exercise 12. Trypan Blue Exclusion and Cell Cytotoxicity

1. Guinea pig complement
2. Fresh serum
3. Hanks Balanced Salt Solution (HBSS)
4. Trypan blue
5. 0.85% NaCl
6. Mouse spleen cells
7. Hemocytometers
8. 37^o C water bath
9. Ice bath
10. Microscopes
11. Microcentrifuge tubes, 5 ml tubes Pipets, tips

Method:

1. Determine the number of cells in the stock sample provided by diluting 1:100, (take 5 µl of sample and add to 495 µl of 0.85% NaCl) then using a hemocytometer as in Exercise 10.  Adjust the cell number so that your stock has 1 x 10⁷ cells/ml.
2. Label 6 tubes by numbering them from 1 to 6. Tube number 1 should contain 400 µl of serum.
3. Add 300 µl of HBSS to tubes 2 through 6. Transfer 100 µl of serum from tube 1 to tube 2. Continue the 4-fold dilution to tube 5. Discard 100 µl from tube 5. Tube 6 is your control tube containing no serum.
4. Transfer 100 µl of cells to each tube from a stock suspension containing 1 x 10⁷ cells/ml.
5. Add 100 µl of guinea pig complement (1/10 dilution) to each tube.
6. Incubate for 1 hour in a 37^o C water bath.
7. Remove the tubes and place them on ice.
8. Add 100 µl of trypan blue to the tubes, in a staggered fashion, so that the trypan blue is not in contact with the cells for longer than 5 min.
9. Count the cells using a hemocytometer distinguishing between cells that have absorbed the dye from those that have not.
10. Report your findings.

1. What is the total number of cells in the original stock solution?
2. What is the total number of viable cells in the original stock solution?
3. Plot the activity of the serum as percent cytotoxicity vs serum dilution.

Exercise 13. Isolation of Whole Mononuclear Cells From Blood