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Telomere length (TL) measurement using flowFISH on proliferating T lymphocytes depends on fixation-permeabilization and RNA nuclease treatment. PBMC from healthy adults were either stimulated with immobilized anti-CD3 and anti-CD28 for 4 days or were cryopreserved on day 0 and thawed for processing on day 4; CD3 + T cells were magnetically sorted from both samples on day 4. Each sample was divided for flowFISH TL analysis and telomere restriction fragment (TRF) Southern blotting. (A) FlowFISH analysis using 3 different pre-hybridization conditions: without fixation-permeabilization prior to probe hybridization (no fix/perm), with fixation-permeabilization (Fix/Perm) prior to hybridization, and with fixation-permeabilization followed by RNase treatment prior to hybridization (Fix/Perm +RNase). Statistical comparisons were done using unpaired t -test, *** p<0.001, and ns=not significantly different. (B) TRF Southern blot analysis. DNA was extracted from CD3+ T cells isolated ex vivo or after stimulation with anti CD3+CD28 for four days and subject to Southern Blot Analysis. TRF lengths are shown at the bottom of both lanes and are the average of three separate 20 pixel-wide analyses <t>using</t> <t>MatLab</t> software running the <t>MaTelo</t> macro (see Methods).
Matlab Macro Matelo, supplied by MathWorks Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Telomere length (TL) measurement using flowFISH on proliferating T lymphocytes depends on fixation-permeabilization and RNA nuclease treatment. PBMC from healthy adults were either stimulated with immobilized anti-CD3 and anti-CD28 for 4 days or were cryopreserved on day 0 and thawed for processing on day 4; CD3 + T cells were magnetically sorted from both samples on day 4. Each sample was divided for flowFISH TL analysis and telomere restriction fragment (TRF) Southern blotting. (A) FlowFISH analysis using 3 different pre-hybridization conditions: without fixation-permeabilization prior to probe hybridization (no fix/perm), with fixation-permeabilization (Fix/Perm) prior to hybridization, and with fixation-permeabilization followed by RNase treatment prior to hybridization (Fix/Perm +RNase). Statistical comparisons were done using unpaired t -test, *** p<0.001, and ns=not significantly different. (B) TRF Southern blot analysis. DNA was extracted from CD3+ T cells isolated ex vivo or after stimulation with anti CD3+CD28 for four days and subject to Southern Blot Analysis. TRF lengths are shown at the bottom of both lanes and are the average of three separate 20 pixel-wide analyses using MatLab software running the MaTelo macro (see Methods).

Journal: Immunity & Ageing : I & A

Article Title: Telomere length dynamics in human memory T cells specific for viruses causing acute or latent infections

doi: 10.1186/1742-4933-10-37

Figure Lengend Snippet: Telomere length (TL) measurement using flowFISH on proliferating T lymphocytes depends on fixation-permeabilization and RNA nuclease treatment. PBMC from healthy adults were either stimulated with immobilized anti-CD3 and anti-CD28 for 4 days or were cryopreserved on day 0 and thawed for processing on day 4; CD3 + T cells were magnetically sorted from both samples on day 4. Each sample was divided for flowFISH TL analysis and telomere restriction fragment (TRF) Southern blotting. (A) FlowFISH analysis using 3 different pre-hybridization conditions: without fixation-permeabilization prior to probe hybridization (no fix/perm), with fixation-permeabilization (Fix/Perm) prior to hybridization, and with fixation-permeabilization followed by RNase treatment prior to hybridization (Fix/Perm +RNase). Statistical comparisons were done using unpaired t -test, *** p<0.001, and ns=not significantly different. (B) TRF Southern blot analysis. DNA was extracted from CD3+ T cells isolated ex vivo or after stimulation with anti CD3+CD28 for four days and subject to Southern Blot Analysis. TRF lengths are shown at the bottom of both lanes and are the average of three separate 20 pixel-wide analyses using MatLab software running the MaTelo macro (see Methods).

Article Snippet: The resulting scanned image was analyzed with the MatLab (MathWorks) macro MATELO ( http://md.technion.ac.il/lecturers/lecturer_desc.asp?lecturerID=10&departmentID=1&contentCatID=4 ) [ ].

Techniques: Southern Blot, Hybridization, Isolation, Ex Vivo, Software