Assessment of the purity and characteristics of rat splenic T cells isolated by one-step discontinuous gradient of percoll
T cells isolated from spleen lymphocytes of rat (Rattus rattus) have been commonly used in an immunological research. However the T cells are still contaminated with other splenic cells. Therefore a simple and reliable technique to isolate the T cells in one step process is needed. The aim of the pr...
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ftdoajarticles:oai:doaj.org/article:5e2d110598754b7b849f5fa521e4683e 2023-05-15T18:05:44+02:00 Assessment of the purity and characteristics of rat splenic T cells isolated by one-step discontinuous gradient of percoll Sulaiman Ngongu Depamede Depamede 2010-06-01T00:00:00Z https://doaj.org/article/5e2d110598754b7b849f5fa521e4683e EN ID eng ind Pusat Penelitian dan Pengembangan Peternakan http://medpub.litbang.pertanian.go.id/index.php/jitv/article/view/653/662 https://doaj.org/toc/0853-7380 https://doaj.org/toc/2252-696X 0853-7380 2252-696X https://doaj.org/article/5e2d110598754b7b849f5fa521e4683e Jurnal Ilmu Ternak dan Veteriner, Vol 15, Iss 2, Pp 157-161 (2010) Percoll Lymphocyte T Cell Rat Mitogen Agriculture S Animal culture SF1-1100 article 2010 ftdoajarticles 2022-12-31T10:07:09Z T cells isolated from spleen lymphocytes of rat (Rattus rattus) have been commonly used in an immunological research. However the T cells are still contaminated with other splenic cells. Therefore a simple and reliable technique to isolate the T cells in one step process is needed. The aim of the present study was to assess whether T cells isolated from spleen lymphocytes of rat as a model by means of discontinuous density gradient of Percoll could give pure T cells with their activities as needed in an immunological research. Isolation was carried out in 5 density gradients of Percoll i.e. 1.052, 1.063, 1.075, 1.085, and 1.122 g/ml. The purity of the cell fractions was determined by immunocytochemistry and fluorescence-activated cell sorting (FACS) methods. The activity of each lymphocyte fraction was determined through cell proliferation assays by culturing the cells in the presence of phytohemaglutinin (PHA) and Concanavalin A (Con A) as T cells inducers. The FACS results show that fraction I and II were found to contain mostly B cells and macrophages. The fractions were obtained on the layers of gradient 1.052/1.063 and 1.063/1.075. Fraction III (gradient 1.075/1.085) contain T cells (43.2%) which consisted of CD4 (34.4%) and CD8 (18.6%), B cells (36.5%), and macrophages (10.0%). Fraction IV was strongly expressed T cell receptors (76.2%) which consisted of CD4 (63.6%) and CD 8 (20.0%), while B cells were found 20.0% and macrophages just about 0.6%. The activity of T cells as expressed by their respond to PHA and Con A shown that fraction III gave the most positive response, while fraction IV which contained the most ‘pure’ T cells gave less activity. From the present study it can be concluded that the discontinuous density gradient of Percoll method can be used to isolate and purify T cells in one step process. Furthermore, the present study indicated that T cells to proliferate in response to mitogen require other immune cells such as B cells and macrophages. Article in Journal/Newspaper Rattus rattus Directory of Open Access Journals: DOAJ Articles |
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Directory of Open Access Journals: DOAJ Articles |
op_collection_id |
ftdoajarticles |
language |
English Indonesian |
topic |
Percoll Lymphocyte T Cell Rat Mitogen Agriculture S Animal culture SF1-1100 |
spellingShingle |
Percoll Lymphocyte T Cell Rat Mitogen Agriculture S Animal culture SF1-1100 Sulaiman Ngongu Depamede Depamede Assessment of the purity and characteristics of rat splenic T cells isolated by one-step discontinuous gradient of percoll |
topic_facet |
Percoll Lymphocyte T Cell Rat Mitogen Agriculture S Animal culture SF1-1100 |
description |
T cells isolated from spleen lymphocytes of rat (Rattus rattus) have been commonly used in an immunological research. However the T cells are still contaminated with other splenic cells. Therefore a simple and reliable technique to isolate the T cells in one step process is needed. The aim of the present study was to assess whether T cells isolated from spleen lymphocytes of rat as a model by means of discontinuous density gradient of Percoll could give pure T cells with their activities as needed in an immunological research. Isolation was carried out in 5 density gradients of Percoll i.e. 1.052, 1.063, 1.075, 1.085, and 1.122 g/ml. The purity of the cell fractions was determined by immunocytochemistry and fluorescence-activated cell sorting (FACS) methods. The activity of each lymphocyte fraction was determined through cell proliferation assays by culturing the cells in the presence of phytohemaglutinin (PHA) and Concanavalin A (Con A) as T cells inducers. The FACS results show that fraction I and II were found to contain mostly B cells and macrophages. The fractions were obtained on the layers of gradient 1.052/1.063 and 1.063/1.075. Fraction III (gradient 1.075/1.085) contain T cells (43.2%) which consisted of CD4 (34.4%) and CD8 (18.6%), B cells (36.5%), and macrophages (10.0%). Fraction IV was strongly expressed T cell receptors (76.2%) which consisted of CD4 (63.6%) and CD 8 (20.0%), while B cells were found 20.0% and macrophages just about 0.6%. The activity of T cells as expressed by their respond to PHA and Con A shown that fraction III gave the most positive response, while fraction IV which contained the most ‘pure’ T cells gave less activity. From the present study it can be concluded that the discontinuous density gradient of Percoll method can be used to isolate and purify T cells in one step process. Furthermore, the present study indicated that T cells to proliferate in response to mitogen require other immune cells such as B cells and macrophages. |
format |
Article in Journal/Newspaper |
author |
Sulaiman Ngongu Depamede Depamede |
author_facet |
Sulaiman Ngongu Depamede Depamede |
author_sort |
Sulaiman Ngongu Depamede Depamede |
title |
Assessment of the purity and characteristics of rat splenic T cells isolated by one-step discontinuous gradient of percoll |
title_short |
Assessment of the purity and characteristics of rat splenic T cells isolated by one-step discontinuous gradient of percoll |
title_full |
Assessment of the purity and characteristics of rat splenic T cells isolated by one-step discontinuous gradient of percoll |
title_fullStr |
Assessment of the purity and characteristics of rat splenic T cells isolated by one-step discontinuous gradient of percoll |
title_full_unstemmed |
Assessment of the purity and characteristics of rat splenic T cells isolated by one-step discontinuous gradient of percoll |
title_sort |
assessment of the purity and characteristics of rat splenic t cells isolated by one-step discontinuous gradient of percoll |
publisher |
Pusat Penelitian dan Pengembangan Peternakan |
publishDate |
2010 |
url |
https://doaj.org/article/5e2d110598754b7b849f5fa521e4683e |
genre |
Rattus rattus |
genre_facet |
Rattus rattus |
op_source |
Jurnal Ilmu Ternak dan Veteriner, Vol 15, Iss 2, Pp 157-161 (2010) |
op_relation |
http://medpub.litbang.pertanian.go.id/index.php/jitv/article/view/653/662 https://doaj.org/toc/0853-7380 https://doaj.org/toc/2252-696X 0853-7380 2252-696X https://doaj.org/article/5e2d110598754b7b849f5fa521e4683e |
_version_ |
1766177242232127488 |