Supplementary Materials Physique?S1. are of central importance in studies of male reproductive biology. The traditional histomorphometric and immunohistochemical methods remain the gold standard in studying the complex dynamics of the testicular tissue. Through past years advances have been made in the application of flow cytometry for the rapid analysis of testicular cell populations. Detection of DNA content and of surface antigens and fluorescent reporters have been widely used to analyze and sort cells. Detection of intracellular antigens can broaden the possibilities Thiostrepton of applying flow cytometry in studies of male reproduction. Here, we Thiostrepton report a detailed protocol for the preparation of rat testicular tissue for detection of intracellular antigens by flow cytometry, and a pipeline for subsequent data analysis and troubleshooting. Rat testicular ontogenesis was chosen as the experimental model to validate the performance of the assay using vimentin and H2AX as intracellular markers for the somatic and spermatogenic cells, respectively. The results show that the assay is reproducible and recapitulates the rat BMP2 testis ontogenesis. carefully. Aliquot approximately 10?mg of tissue per each sample using the McPhersonCVannas scissors. Record the exact weights of the tissue aliquots. 1.3 Mince the testicular tissue with the McPhersonCVannas scissors in 200?L of the digestion medium until all seminiferous tubule pieces are 1?mm long. Add the remaining 800?L of the digestion medium to each sample. 1.4 Incubate for 25?min at 37?C with slow continuous rotation. Perform an additional mechanical disruption every 5?min by carefully pipetting the solution five times back and forth using 1000?L Maxymym Recovery tips (Axygen, Corning Inc., Corning, NY, USA). Performing this step according to instructions is critical to ensure an optimal recovery of the fragile spermatocytes and to minimize debris. 1.5 Filter the cell suspension using a 35?m pore size to achieve single cell suspension. A flow cytometry tube cell strainer cap (cat. # 352235; BD Falcon, BD Biosciences, Franklin Lakes, NJ, USA) attached to an Eppendorf tube functions well Thiostrepton for the filtration. Pipette the cell suspension Thiostrepton slowly through the Thiostrepton mesh to avoid clogging and cell damage. Pellet the cells by centrifugation (400? em g /em , 10?min at +4?C), discard the supernatant and resuspend the pellet in 1?mL of PBS. If preparing samples without dead cell discrimination, continue to step 3 3.1. Dead cell discrimination em (optional) /em A fixable dead cell stain can be added to the cell suspension. We used LIVE/DEAD Near\IR (Invitrogen, Thermo Fischer Scientific, Waltham, MA, USA), which binds to amines on cell surface, but also intracellularly when the integrity of the cell is compromised. Intracellular amine staining yields an intense fluorescent signal in flow cytometry and allows the exclusion of dead cells from the downstream analyses. 2.1 Add the LIVE/DEAD Near\IR stain reconstituted in DMSO to the cells at a final concentration of 1 1?:?10?000. Incubate for 30?min on ice and pellet by centrifugation (400? em g /em , 10?min at +4?C) and resuspend in 1?mL of PBS. Fixation and permeabilizationTo allow the detection of intracellular antigens by specific antibodies and to enable long\term storage of the samples, the testicular cells are fixed using 4% paraformaldehyde (PFA) and permeabilized with 90% methanol (an adaptation of the protocol from Cell Signaling Technology Inc., Danvers, MA, USA). 3.1 Add 32% PFA (cat. # 15714; Electron Microscopy Sciences, Hatfield, PA, USA) to a final concentration of 4% to the cell suspension while vortexing the solution slowly. This prevents aggregation of the cells during fixation. For fixation, incubate the samples for 10?min at 37?C with gentle rotation. Chill the samples on ice for 1?min. 3.2 Centrifuge the cells at 1200? em g /em , 7?min at 4?C to remove the fixative. Discard the supernatant and resuspend the pellet in 100?L of PBS. Permeabilize the cells by adding 900?L of ice\cold 100% methanol in a drop\wise fashion while gently vortexing the cells. Incubate for 30?min on ice. After this step, the samples can be stored in ?20?C. Detection of intracellular antigens and DNA content of the testicular cells 4.1 Remove methanol solution from the samples by centrifugation at 1200? em g /em , 7?min at 4?C. Discard the supernatant. For washing resuspend the pellet in with 1?mL of incubation buffer (0.5% sera of choice in PBS). If using.

Supplementary Materials Physique?S1