Spleens of mice immunized with sheep cells were taken and spleen cell suspension was made, which was combined with a certain amount of indicator cells (sheep cells) and complement, injected into a homemade chamber, and incubated at 37°C. After incubation, individual scattered antibodies formed to release antibodies, which bound to the surrounding sheep erythrocytes (antigens) and lysed the sheep cells with the participation of complement, resulting in formation of a round, transparent, visible to the naked eye, around the antibody-forming cells. Hemolytic zone - hemolytic vacuoles.
Operation method
Detection of antibody-producing cells
Materials and Instruments
Healthy mice Move 1. Immunized animalsOne milliliter of 25% sheep erythrocyte suspension was aseptically injected into the peritoneal cavity of mice. Caveat Splenocyte suspension preparation should be done in an ice water bath. For more product details, please visit Aladdin Scientific website.
Sheep Erythrocyte Suspension Hanks Liquid Petroleum Jelly Oil
Syringes, flatware, funnel, gauze, mortar and pestle
2. Preparation of splenic cytosol
(1) After 4 days of immunization, mice were executed by cervical dislocation, and the spleen was removed, adipose tissue was removed, placed in a flat dish, and washed 1~2 times with cold Hanks solution.
(2) Remove the spleen and crush it in a mortar, add 2~3 ml of Hanks' solution, and blow repeatedly with a pipette several times to disperse the cells. This cell suspension was filtered through 4~8 layers of gauze, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded.If there are too many cells, add 4 ml of distilled water to mix quickly, half a minute later, immediately add an equal amount of Hanks solution to mix 1000 rpm centrifugation for 10 minutes, discard the supernatant.
(3) The precipitated cells were washed with Hanks' solution 1~2 times, and the supernatant was discarded, then Hanks' solution was added to make the total amount reach 5 ml. Blow with a nipple pipette to mix the precipitated cells, which are 50-fold diluted splenocytes.
(4) Take 0.1 ml of this suspension and put it into another small test tube and then add 0.9 ml of Hanks solution to make a 500-fold dilution of spleen cell suspension.3. Production of glass chambersTake a clean (oil-free) slide, stick three strips of transparent tape according to the figure below, apply a thin layer of petroleum jelly on the tape (do not apply it to the small chamber), and then use tweezers to take two cover slips and place them on it to make two small chambers. Use the end of the tweezers to flatten the cover piece firmly (to maintain a certain volume of the chambers). Seal the bottom end of the cover slips (either of them) with petroleum jelly, and the top end serves as an injection of the cell mixture. 
Fig. 1 Schematic diagram of the glass chamber4. Preparation of Cell MixTake a small test tube, use a 1 ml pipette to draw 0.2 ml of the indicated cell suspension, use another pipette to draw 0.2 ml of 500-fold diluted splenocyte suspension, and mix it well to be the examined cell mixture suspension.
5. Perfusion of mixed cellsAspirate the mixed suspension of the examined cells with a 100 ul microsampler, gently fill the chamber with liquid at the open end of the chamber (do not make air bubbles, do not let the liquid spill out), and record the number of microliters of cell suspension actually injected (about 70 ul).For each sample, inject 2 chambers and take the average value.
6. Use a toothpick to seal the opening of the chamber with a small amount of petroleum jelly.
7. Place the prepared specimen horizontally in a wet box and incubate at 37°C for 60 minutes.
8. Empty spot count for hemolysisObserve the vacuoles with the naked eye and count the number of hemolytic vacuoles present in both chambers. Blurred empty spots can be examined under low magnification; a true hemolytic empty spot must have a lymphocyte in the center surrounded by a clear area.The total number of antibody-producing cells in the whole spleen can be calculated according to the following formula: 