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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fticardojs:oai:ojs.pkp.sfu.ca:article/653 2023-07-02T03:33:36+02:00 Assessment of the purity and characteristics of rat splenic T cells isolated by one-step discontinuous gradient of percoll Depamede, Sulaiman Ngongu Depamede 2012-03-04 application/pdf http://medpub.litbang.pertanian.go.id/index.php/jitv/article/view/653 https://doi.org/10.14334/jitv.v15i2.653 eng eng Indonesian Center for Animal Research and Development (ICARD) http://medpub.litbang.pertanian.go.id/index.php/jitv/article/view/653/662 http://medpub.litbang.pertanian.go.id/index.php/jitv/article/view/653 doi:10.14334/jitv.v15i2.653 Copyright (c) 1970 Indonesian Journal of Animal and Veterinary Sciences http://creativecommons.org/licenses/by/4.0 Jurnal Ilmu Ternak dan Veteriner; Vol 15, No 2 (2010): JUNE 2010; p.157-161 2252-696X 0853-7380 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Peer-reviewed Article 2012 fticardojs https://doi.org/10.14334/jitv.v15i2.653 2023-06-12T18:41:14Z 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. Key words: Percoll, Lymphocyte, T Cell, Rat, ... Article in Journal/Newspaper Rattus rattus Indonesian Center for Animal Research and Development : Scientific Journal of ICARD |
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Indonesian Center for Animal Research and Development : Scientific Journal of ICARD |
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fticardojs |
language |
English |
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. Key words: Percoll, Lymphocyte, T Cell, Rat, ... |
format |
Article in Journal/Newspaper |
author |
Depamede, Sulaiman Ngongu Depamede |
spellingShingle |
Depamede, Sulaiman Ngongu Depamede Assessment of the purity and characteristics of rat splenic T cells isolated by one-step discontinuous gradient of percoll |
author_facet |
Depamede, Sulaiman Ngongu Depamede |
author_sort |
Depamede, Sulaiman Ngongu 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 |
Indonesian Center for Animal Research and Development (ICARD) |
publishDate |
2012 |
url |
http://medpub.litbang.pertanian.go.id/index.php/jitv/article/view/653 https://doi.org/10.14334/jitv.v15i2.653 |
genre |
Rattus rattus |
genre_facet |
Rattus rattus |
op_source |
Jurnal Ilmu Ternak dan Veteriner; Vol 15, No 2 (2010): JUNE 2010; p.157-161 2252-696X 0853-7380 |
op_relation |
http://medpub.litbang.pertanian.go.id/index.php/jitv/article/view/653/662 http://medpub.litbang.pertanian.go.id/index.php/jitv/article/view/653 doi:10.14334/jitv.v15i2.653 |
op_rights |
Copyright (c) 1970 Indonesian Journal of Animal and Veterinary Sciences http://creativecommons.org/licenses/by/4.0 |
op_doi |
https://doi.org/10.14334/jitv.v15i2.653 |
_version_ |
1770273621853339648 |