CHINHOYI UNIVERSITY OF TECHNOLOGY

SCHOOL OF HEALTH SCIENCES AND TECHNOLOGY

DEPARTMENT OF BIOTECHNOLOGY
NAME : TOGA BRANDON
REG NUMBER : C22151745G
PROGRAM : BIOTECHNOLOGY ( BSBIO )
MODULE : ENZYMOLOGY AND IMMUNOLOGY
LECTURER : MR MAZADZA
LEVEL : 1.2
ASSIGNMENT : 3
Assignment 3
Describe an experiment you would use in the isolation and enumeration of lymphocytes
Isolation and enumeration of lymphocytes can be using a method called the density gradient
centrifugation. Centrifugation is a commonly used method for blood processing as it can easily
fraction blood into different components without convection through differential centrifugation
and selective removal (Saunders and Britton,2019). Aquas media together with centrifugation has
been used to separate and isolate blood cells types. (Alenkin et al,2014). The following
procedures are done during centrifugation.
1. Firstly, about 20ml of blood sample is collected from the patient’s vein,
2. The collected blood sample is transferred to four 7ml heparinized green top tubes in total
20mls,
3. The blood from the heparinised tubes is further poured together into one 50ml blue cap tube,
4. The whole blood is diluted to make 1:1 with 1x PBS,
5. Then by carefully underlaying 10ml of histopaque-1077, 20ml of diluted blood is transferred
to a 50ml tube,
6. Immediately, the blood is centrifugated at 1800rpm (700g) for 30 minutes in a cooling
centrifuge (4 degrees Celsius),
7. The tube is carefully removed from the centrifuge, observing the three layers, top layer is
clearly supernatant which contains plasma, middle is opaque fluid containing the PBMC and the
bottom layer is erythrocyte layer i.e RBC,
8. The supernatant within 15ml of the opaque layer of the PBMC is carefully removed and
discarded,
9. The buffy coat layer of PBMC (around 5ml) is quickly transferred into new 50ml tube.
10. 1x PBS is added to the PBMC suspension to obtain a final volume of 45ml, the mixture is
centrifugated at 1800rpm (700g) for 10minutes in a cooling centrifuge or incubator,
11. The supernatant is poured off and 1XPBS combined to obtain the final volume 30ml, the
mixture is centrifugated at 1500rpm (500g) for 10min in a cooling centrifuge (4 degrees Celsius)
or incubator to remove platelets,
12. The supernatant is poured off once again where as the pellet in 5ml of the freezing medium
(90%FBS+10%DMSO filter sterilised) is resuspended,
13. Then aliquot 1ml of cell suspension per vial (cryogenic vial) and store at 80degree Celsius
overnight and then transfer in the liquid nitrogen cryofreezer.
After 24 hours of growth, it may be necessary to add 15-20 mL of fresh media and transfer to a
larger flask.
The lymphocytes can be grown for up to 4 days and the Results are obtained by flow cytometry
technique, which involves the use of fluorochromes, this method uses light energy from a laser at
a given wavelength (Freeman, 2013). The technique provides accurate results and purifies small
and complex subpopulations.
Culture in the presence of either IL-2 or IL-15, T lymphocytes make up more than 98% of cells,
as determined by positive CD3 staining as well as positive CD4 and/or CD8 staining. (Abbas and
Litchman,2016)

References
Alenkin D, Yermekbayeva L, Mujib S, Vesterberg A, Newman E, Yamazaki K, Cossar D and
Dhe-Paganon S: A centrifugation-free high-throughput protein purification system, Journal of
Medical Science, vol. 3, no. 1, pp. 55-56, 2014
Abbas E and Litchman H, Cellular and Molecular Immunology 7th Ed, 2016
Freeman W.H; Kuby Immunology 8th Edition, 2013
Kuma R, Robbins and Cotran G, Pathologic Basis of Diseases, 2014
Saunders R, Britton J, Immunology Cell Biology, (2019) 88: p.1013-1093