Flow Cytometry of Murine Spermatocytes

Valeriya Gaysinskaya1, Alex Bortvin2

1 Department of Biology, Johns Hopkins University, Baltimore, Maryland, 2 Department of Embryology, Carnegie Institution for Science, Baltimore, Maryland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.44
DOI:  10.1002/0471142956.cy0744s72
Online Posting Date:  April, 2015
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Abstract

Protocols for purification of murine male germ cells by FACS based on Hoechst 33342 (Ho342) dye staining have been reported and optimized. However, the protocols are often challenging to follow, partly due to difficulties related to sample preparation, instrument parameters, data display, and selection strategies. In addition, troubleshooting of flow cytometry experiments usually requires some fluency in technical principles and instrument specifications and settings. This unit describes setup and procedures for analysis and sorting of male meiotic prophase I (MPI) cells and other germ cells. Included are procedures that guide data acquisition, display, gating, and back‐gating critical for optimal data visualization and cell sorting. Additionally, a flow cytometry analysis of spermatogenesis‐defective testis is provided to illustrate the applicability of the technique to the characterization and purification of cells from mutant testis. © 2015 by John Wiley & Sons, Inc.

Keywords: adult mouse testis; Hoechst 33342; meiotic prophase I; flow cytometry; gating; cell sorting

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Optimization of PMT Voltage Using Control Cells
  • Basic Protocol 2: Setup of Working Flow Cytometry Profile For Ho342/PI‐Stained Testicular Cells
  • Basic Protocol 3: Setup of Experimental Gates and Sorting of Wild‐Type Testicular Subpopulations
  • Basic Protocol 4: Flow Cytometry Analysis of Spermatogenesis‐Defective Testis Using Ho342/PI Staining
  • Support Protocol 1: Preparation of Stained Single‐Cell Suspensions from Adult Murine Testis
  • Support Protocol 2: Analysis of Purity of Sorted Cells by Immunofluorescence on Nuclear Spreads
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Optimization of PMT Voltage Using Control Cells

  Materials
  • Control samples (see protocol 5)
    • Unstained cells
    • Ho342‐stained cells
    • PI‐stained cells
    • Ho342/PI‐stained cells
  • FACS cytometer (BD FACSAria III or similar) equipped with:
    • Excitation optics: near‐UV (375‐nm) and blue (488‐nm) lasers
    • Detection optics: filter combination consisting of 450/40 bandpass (Ho342‐blue detection), 670 longpass (Ho342‐red and PI detection), and 610 dichroic longpass mirror (beam splitter)
  • FACS analysis software (BD FACSDiva or similar)
NOTE: See Table 7.44.1 for fluorochrome specifications and Table 7.44.2 for laser and optics device configuration.
Table 7.4.1   MaterialsFluorochrome SpecificationsLaser and Optics Device Configuration

Fluorochrome Excitation/emission max (nm) Comment
Hoechst 33342 (Ho342) 350/461 max (bound to DNA) Wide emission spectrum allows Ho342 red detection
Propidium iodide 540/607 (bound to DNA)
Laser and optics device PMTs Longpass mirrors Bandpass filters Fluorochromes
Blue 488‐nm Trigon A 655LP 695/40 N/A here; otherwise PerCP‐Cy5.5, PerCP
B 502LP 530/30 N/A here; otherwise FITC
C 488/10 SSC
Red 633‐nm Trigon A 735LP 780/60 N/A here; otherwise, APC‐Cy7, APC‐H7
B 660/20 N/A here; otherwise, APC
Near UV 375‐nm A 610LP 670LP Ho342‐red, propidium iodide
Octagon B 450/40 Ho342‐blue; otherwise, DAPI
C 502LP 510/50 N/A here; otherwise, AmCyan

Table 7.4.2   MaterialsFluorochrome SpecificationsLaser and Optics Device Configuration

Fluorochrome Excitation/emission max (nm) Comment
Hoechst 33342 (Ho342) 350/461 max (bound to DNA) Wide emission spectrum allows Ho342 red detection
Propidium iodide 540/607 (bound to DNA)
Laser and optics device PMTs Longpass mirrors Bandpass filters Fluorochromes
Blue 488‐nm Trigon A 655LP 695/40 N/A here; otherwise PerCP‐Cy5.5, PerCP
B 502LP 530/30 N/A here; otherwise FITC
C 488/10 SSC
Red 633‐nm Trigon A 735LP 780/60 N/A here; otherwise, APC‐Cy7, APC‐H7
B 660/20 N/A here; otherwise, APC
Near UV 375‐nm A 610LP 670LP Ho342‐red, propidium iodide
Octagon B 450/40 Ho342‐blue; otherwise, DAPI
C 502LP 510/50 N/A here; otherwise, AmCyan

Basic Protocol 2: Setup of Working Flow Cytometry Profile For Ho342/PI‐Stained Testicular Cells

  Materials
  • FACS cytometer and analysis software (see protocol 1)
  • Ho342/PI‐stained testicular cell suspension (see protocol 5)

Basic Protocol 3: Setup of Experimental Gates and Sorting of Wild‐Type Testicular Subpopulations

  Materials
  • Ho342/PI‐stained testicular cell suspension (see protocol 5)
  • 5% (v/v) newborn calf serum (NCS) in Gey's balanced salt solution (GBSS; Sigma), filtered through 40‐μm nylon cell strainer
  • 5‐ml round‐bottom polystyrene tubes
  • 35‐μm cell strainer cap (BD Falcon)
  • FACS cytometer and analysis software (see protocol 1)

Basic Protocol 4: Flow Cytometry Analysis of Spermatogenesis‐Defective Testis Using Ho342/PI Staining

  Materials
  • Ho342/PI‐stained Mael−/− testicular cell suspension (see protocol 5)
  • FACS cytometer and analysis software (see protocol 1)

Support Protocol 1: Preparation of Stained Single‐Cell Suspensions from Adult Murine Testis

  Materials
  • Wild‐type male mice (C57BL/6 J), 2‐4 months old (Jackson Laboratory)
  • Mutant male mice (Maelstrom knockout, Mael−/−), 2‐5 months old (Soper et al., )
  • Gey's balanced salt solution (GBSS; Sigma)
  • Collagenase/DNase solution (see recipe), ice cold
  • Trypsin/collagenase/DNase solution (see recipe), prewarmed (35°C)
  • 2.5% (w/v) trypsin (10×) (Gibco)
  • 1 mg/ml DNase I (Invitrogen, cat. no. 18068‐015) in 50% (v/v) glycerol
  • 10 mg/ml Hoechst 33342 (Ho342, aqueous solution; Life Technologies)
  • 0.4% (w/v) trypan blue
  • Newborn calf serum (NCS)
  • 1 mg/ml propidium iodide (PI; Sigma)
  • Dissection tools: scissors, straight and angled fine‐tipped forceps
  • 15‐ml sterile conical tubes (BD Falcon)
  • 35°C shaking water bath or incubator
  • Plastic disposable transfer pipets
  • 100‐ and 40‐μm nylon cell strainers (Falcon)
  • Hemocytometer
  • Phase‐contrast microscope
  • Sterile 5‐ml polypropylene culture tube
  • 12 × 75−mm tube with 35‐μm cell strainer cap (BD Falcon)

Support Protocol 2: Analysis of Purity of Sorted Cells by Immunofluorescence on Nuclear Spreads

  Materials
  • Sorted cells (50 to 100 μl)
  • Hypotonic buffer (see recipe)
  • 100 mM sucrose solution (see recipe)
  • Fixative solution (see recipe), freshly prepared
  • 1× phosphate‐buffered saline (PBS; appendix 2A)
  • 1× PBS/0.05% Triton X‐100
  • Blocking buffer (see recipe)
  • Primary antibodies:
    • 1 mg/ml rabbit anti‐SCP3 (Abcam, cat. no. ab15093)
    • 1 mg/ml mouse anti‐phospho‐histone (yH2AX; Millipore, cat. no. 05‐636)
    • 200 μg/ml mouse anti‐DMRT1 (Santa Cruz Biotechnology, cat. no. H0712)
  • Primary antibody buffer (see recipe)
  • Secondary antibodies:
    • 2 mg/ml donkey anti‐rabbit Alexa Fluor 594 (Life Technologies)
    • 2 mg/ml donkey anti‐mouse Alexa Fluor 488 (Life Technologies)
  • Secondary antibody buffer (see recipe)
  • 5 μg/ml 4,6‐diamidino‐2‐phenylindole (DAPI)
  • Mounting medium for fluorescence (Vectashield)
  • 1.5‐ml microcentrifuge tubes
  • Microscope slides (e.g., SuperFrost Plus; VWR)
  • Humid chamber
  • 42°C hot plate
  • Fluorescence microscope
NOTE: Unless otherwise indicated, all steps are carried out at room temperature.
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Figures

Videos

Literature Cited

Literature Cited
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