A sequential methodology that allows apoptotic cell sorting and FISH analysis in human testicular cells

Figures & data

Figure 1.  Types of annexin V-FITC and propidium iodide staining. Annexin V-FITC and propidium iodide staining enables the identification of apoptotic, necrotic, and viable populations. Their mechanism of action is highlighted. Scale bar represents 10 µm.

Table 1. Results from enzymatic disaggregation and specific apoptotic-/necrotic-staining observed in 13 of 17 patients with optimized protocol. (Four samples were used for the optimization.)

	Cell count	Apoptotic cells (%)	Necrotic cells (%)	Viable cells (%)
446BT	1.8x10^5	11%	40.2%	48.9%
454BT	4.6x10^6	16%	42.2%	39.8%
480BT	1x10^6	16%	30%	54%
486BT	4.4x10^6	15%	27%	58%
487BT	5x10^6	14%	46%	39%
492BT	1.1x10^6	3%	46%	51%
498BT	1.7x10^5	14%	7%	79%
501BT	4.7x10^6	48%	13%	39%
526BT	1.1x10^6	21.1%	0%	78.9%
527BT	2.8x10^5	16%	18%	66%
537BT	6.2x10^5	26.2%	0%	73.8%
538BT	8.9x10^5	39.3%	4.5%	56.2%
554BT	1.3x10^6	12%	12%	75%
Mean	1.9x10^6	19.4%	22%	58.4%
SD	1.9x10^6	12.1%	17.5%	14.9%

Figure 2.  Flow cytometry separation of cells. A) Forward Scatter (FS) / Side Scatter (SS) window. Polygon R1 selects cells from the whole population in order to discard cell fragments (left) and aggregates (right). The subsequent graphs relate to polygon R1. B₁) Non-stained control, whole population without labeling. B₂) PI single-stained control, with the necrotic population in red and the non-stained population in blue. B₃) FITC single-stained control, with the AV-FITC stained population in green and the non-stained population in blue. C) Study population (Annexin V-FITC plus propidium iodide). Apoptotic (R2 in green), viable (R3 in blue), and necrotic (R4 in red) populations are clearly identified. The selected populations correspond to the green (R2) and blue (R3) polygons in C.

Table 2. Flow cytometry results obtained from 3 out of 14 patients with optimized parameters. (Eleven samples were used for the optimization.)

	Apoptotic fraction			Viable fraction
	Percentage	Cell count	Purity	Percentage	Cell count	Purity
537BT	22%	1.1x10^5	77%	53.7%	1.8x10^5	74%
538BT	27.3%	1.5x10^5	72%	42.7%	1.9x10^5	74%
554BT	16.6%	9.3x10^5	62%	40%	2.9x10^5	100%
Mean	22%	4x10^5	70.3%	45.5%	2.2x10^5	82.7%
SD	5.3%	4.6x10^5	7.6%	7.3%	6.2x10^4	15%

Figure 3.  Triple-color FISH. The hybridization of centromeric probes for chromosomes X (green), Y (red), and 18 (blue) to apoptotic and viable cell nuclei are shown. Images A – C are from the apoptotic fraction. Images D – F are from the viable fraction. Scale bar represents 10 µm. (SC: Sertoli Cells; Sg: Spermatogonium; Pp: Prophase; Ss: Secondary Spermatocyte; St: Spermatid; Sz: Spermatozoon).

Figure 4.  Scheme of the sequential methodology. The methodology developed consists 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 (PI: propidium iodide; AV-FITC: Annexin V conjugated with FITC).