PMID- 22988972
OWN - NLM
STAT- MEDLINE
DCOM- 20130419
LR  - 20121115
IS  - 1939-6376 (Electronic)
IS  - 1939-6368 (Linking)
VI  - 58
IP  - 6
DP  - 2012 Dec
TI  - A sequential methodology that allows apoptotic cell sorting and FISH analysis in 
      human testicular cells.
PG  - 354-61
LID - 10.3109/19396368.2012.717163 [doi]
AB  - The objective of this study was to develop a methodology that permits the 
      detection and separation of apoptotic cells in human testicular tissue and their 
      subsequent cytogenetic analysis by fluorescence in situ hybridization (FISH). The 
      sequential methodology consisted of five steps: 1) enzymatic disaggregation of 
      testicular tissue, 2) specific staining of apoptotic cells, 3) cell sorting by 
      flow cytometry, 4) cell fixation, and 5) FISH. Enzymatic disaggregation yielded 
      cell counts that ranged from 1.7x10(5) to 5x10(6) cells, and viability values 
      greater than 72%. The apoptotic (mean +/- SD: 22% +/- 5.3%) and viable (45.5% +/- 7.3%) 
      populations were identified and selected by flow cytometry and demonstrated 
      purity values ranging between 62% and 100%. The paraformaldehyde fixation of the 
      selected fractions resulted in cell loss values of less than 10%. The application 
      of three treatments before FISH (membrane permeabilization, elimination of 
      cytoplasmic components, and re-fixation of the sample) resulted in hybridization 
      frequencies of greater than 98%. In both selected fractions, cells of all 
      spermatogenic stages and Sertoli cells were identified. The methodology developed 
      has enabled the preparation of a cellular suspension with optimal viability and 
      counting, the efficient selection of the apoptotic population, and its analysis 
      by cytogenetic techniques. The application of this methodology in testicular 
      cells should help establish whether there is a direct relationship between 
      chromosome anomalies and apoptosis.
FAU - Garcia-Quevedo, Lydia
AU  - Garcia-Quevedo L
AD  - Unitat de Biologia Cel.lular, Facultat de Biociencies, Universitat Autonoma de 
      Barcelona, 08193-Bellaterra (Cerdanyola del Valles), Spain.
FAU - Sarrate, Zaida
AU  - Sarrate Z
FAU - Vidal, Francesca
AU  - Vidal F
FAU - Blanco, Joan
AU  - Blanco J
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20120918
PL  - England
TA  - Syst Biol Reprod Med
JT  - Systems biology in reproductive medicine
JID - 101464963
SB  - IM
MH  - *Apoptosis
MH  - Cell Separation/*methods
MH  - Flow Cytometry
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Male
MH  - Testis/*cytology
EDAT- 2012/09/20 06:00
MHDA- 2013/04/23 06:00
CRDT- 2012/09/20 06:00
PHST- 2012/09/20 06:00 [entrez]
PHST- 2012/09/20 06:00 [pubmed]
PHST- 2013/04/23 06:00 [medline]
AID - 10.3109/19396368.2012.717163 [doi]
PST - ppublish
SO  - Syst Biol Reprod Med. 2012 Dec;58(6):354-61. doi: 10.3109/19396368.2012.717163. 
      Epub 2012 Sep 18.