Comparison of different clearing and acquisition methods for 3D imaging of murine intestinal organoids
2Université de Paris, Imagine Institute, Laboratory of Intestinal Immunity, INSERM UMR1163, Paris 75015, France
MATERIALS AND METHODS
- LWRN cells: ATCC® CRL3276™ (from Dr. T. Stapenbeck, Washington University School of Medicine, St Louis, MO, USA)
- DMEM, High Glucose, GlutaMAXTMSupplement: GibcoTM-Thermo Fisher FR (Cat. # 61965240)
- DMEM/F12: GibcoTM-Thermo Fisher FR (Cat. # 11320033)
- Advanced DMEM/F12: GibcoTM-Thermo Fisher FR (Cat. # 12634028)
- Penicillin/Streptomycin: GibcoTM-Thermo Fisher FR (Cat. # 15140122)
- Foetal Bovine Serum (FBS): GibcoTM-Thermo Fisher FR
- Geneticin G418: GibcoTM-Thermo Fisher FR (Cat. # 10131035)
- Hygromycin: Sigma-Aldrich FR (Cat. # H7772)
- Glutamine: GibcoTM-Thermo Fisher FR (Cat. # A2916801)
- TriplExpress: GibcoTM-Thermo Fisher FR (Cat. # 12605036)
- Matrigel®: Corning (Cat. # 354230)
- Phosphate-Buffered Saline without calcium or magnesium (DPBS): GibcoTM-Thermo Fisher FR (Cat. # 14190-144)
- 1.4Dithiotreiol (DTT): Roche FR (Cat. # 10708984001)
- Ethylenediaminetetraacetic acid (EDTA): Sigma-Aldrich FR (Cat. # E6758)
- Paraformaldehyde 36%: Electron microscopy Sciences FR (Cat. # 15714)
- BSA (Bovin Serum Albumin): Sigma-Aldrich FR (Cat. # A7906)
- Triton X-100: Sigma-Aldrich FR (Cat. # 93443)
- Antibody diluent: Life Technologies FR (Cat. # 003218)
- Monoclonal mouse Anti-sucrase-isomaltase antibody: Sigma-Aldrich FR (Cat. # WH0006476M1)
- Goat Anti-Mouse-Alexa-Fluor 488: Abcam FR (Cat. # ab 150113)
- Phalloidin Alexa-Fluor 568: Invitrogen FR (Cat. # A12380)
- NucBlueTM Fixed Cell Stain Ready Probes TM(DAPI): Invitrogen FR (Cat. # R37606)
- RapiClear 1.47: Nikon FR (Cat. # 2SUN0001)
- N-Methyl-D-glucamine: Sigma-Aldrich FR (Cat. # U5378)
- Iohexol (Nicodenz): Proteogenix FR (Cat. # 1002424)
- 2,2′-thiodiethanol: Sigma-Aldrich FR (Cat. # 88561)
- Quadrol (N,N,N′,N′-Tetrakis ((2-Hydroxypropyl)ethylenediamine): Sigma-Aldrich FR (Cat. # 122262)
- Urea: Sigma-Aldrich FR (Cat. # U5378)
- Triton X-100: Sigma-Aldrich FR (Cat. # 93443)
- Sucrose: Sigma-Aldrich FR (Cat. # S7903)
- Triethanolamine: Sigma-Aldrich FR (Cat. # 90279)
- IBIDI® µ-Slide 8 well: IBIDI cells in focus, 80821
- BRAND® cavity slides (L × W76 mm × 26 mm, thickness 1.2–1.5 mm, 1 concavity): Merck, BR475505-50EA
- Ligthsheet Z.1 Capillary: Zeiss product sold with Ligthsheet Z.1 microscope
- Twinsil® Speed picodent: Rotec (Cat. # 1 300 1002)
- Low-melting agarose: Sigma-Aldrich FR (Cat. # 9045)
Intestinal organoids obtaining
Culture medium preparation
1.Day 0: L-WRN cells cultured in 25 ml of “L-Cell medium” (DMEM, High Glucose, GlutaMAXTM Supplement + 10% Foetal Bovine Serum (FBS) + Penicillin/Streptomycin) in 150 cm2 flask.
2.Day 1: Addition of Geneticin G418 (500 µg/ml) and hygromycin (500 µg/ml) to the “L-Cell medium”.
3.Day 5: When the confluence is reached, cells are washed with 20 ml of PBS1X and peeled off with 1 ml of TriplExpress. After detachment, cells are re-suspended in 12 ml of “L-Cell medium”.
1.Addition of 113 ml of “L-Cell Medium” to the 12 ml L-WRN cell suspension, and division into 5 flasks of 150 cm2 (25 ml/flask, at 37°C and 5% CO2).
2.After 4 d, when the confluence is reached, cells are washed with 10 ml of “Primary Culture medium” (Advanced DMEM/F12 + 20%FBS + Penicillin/Streptomycin + Glutamine).
2.1.Addition of 25 ml per vial of “Primary Culture medium” and incubation at 37°C and 5% CO2 during 24 h.
2.2.Medium is collected in 50 ml tubes and 25 ml fresh “Primary Culture medium” is added to each flask.
2.3.Centrifugation of the 50 ml tubes at 2000 g during 5 min and collection of the supernatant in a sterile 1 L bottle (approximately 125 ml). This medium is kept at 4°C.
2.4.Each 24 h, medium from the same flask is collected during 4 d.
2.5.After the fourth collection, addition of an equal volume (500 ml) of “Primary Culture medium” (final concentration: 50%).
2.6.The “L-WRN50% medium” is well mixed, split in several 50 ml tubes and kept at −20°C until use.
Crypts isolation and organoid obtaining
1.Small pieces of SI are put in pre-cooled 10 mM dithiothreitol (DTT) in PBS1X during 10 s and in 8 mM ethylenediaminetetraacetic acid (EDTA) in PBS1X, on ice, during 1 h (stirring vigorously every 15 min).
2.The supernatant is removed and pre-cooled PBS1X is put on small pieces of small intestine and vigorously pipetted using 10 ml pipette to shake tissue fragments.
3.After sedimentation, the supernatant is collected in a clean 50 ml tube and centrifuged at 70 g for 5 min.
4.The pellet is homogenized in 5 ml of DMEM/glutamax and crypts are counted in 20 µl DMEM by light microscopy.
5.Crypts are centrifuged at 200 g during 5 min and the pellet is taken in Matrigel®.
1.Re-suspension of the pellet of crypts in 400 µl Matrigel®, and seeding in a pre-warmed IBIDI® µ-Slide 8-well (50 µl per well).
2.Incubation at 37°C, 5% CO2 during 10 min to allow Matrigel® polymerization.
3.Addition of 300 µl conditioned LWRN-50% medium.
4.Fresh medium is changed every 3 d and organoids are passed every 7 d.
Immunostaining and clearing
1.Enterocytes borders staining.
1.1.Monoclonal mouse Anti-sucrase-isomaltase antibody (dilution 1/100) in antibody diluent at 37°C during 1 h.
1.2.Washing: PBS1X + 0.1% BSA, at room temperature, 2 × 5 min.
1.3.GoatAnti-Mouse-AF488 secondary antibody (dilution 1/100) in antibody diluent at 37°C during 30 min.
1.4.Washing: PBS1X + 0.1% BSA, at room temperature, 2 × 5 min.
2.1.Phalloidin-AF568 (concentration 6 µM) in antibody diluent at 37°C during 30 min.
2.2.Washing: PBS1X + 0.1% BSA, at room temperature, 2 × 5 min.
3.1.DAPI: NucBlueTM (dilution 2 drops/ml) in PBS1X, at room temperature during 5 min.
3.2.Washing: PBS1X at room temperature, 2 × 5 min.
1.After immunostaining: 30%, 60%, 80% TDE in PBS1X, at room temperature during 1 d each.
2.Keep in 80% TDE until imaging.
1.After fixation: Reagent-1 (Mixture of Urea (25 wt% final concentration), Quadrol (25 wt% final concentration), Triton X100 (15 wt% final concentration) and dH2O (from Susaki et al., 2015 , at 37°C during 4 h.
2.Washing: PBS1X, at room temperature under low agitation: 2 h, overnight, 2 h.
4.Reagent-2 (Mixture of Urea (25 wt% final concentration), Sucrose (50 wt% final concentration), Triethanolamine (10 wt% final concentration) and dH2O (from Susaki et al., 2015 , at 37°C during 4 h and then at room temperature until imaging.
1.For non-cleared samples, the microscope imaging chamber was filled with 20 ml of water and acquisitions were done using 10X illumination objectives and Plan Aprochromat 20X water immersion objective (NA 1.0).
2.For samples cleared with RapiClear and TDE, the microscope imaging chamber was filled with 20 ml of 80% TDE (refractive index ≈ 1.47) and acquisitions were done using LSFM 10X illumination objectives and Clr PlanNEOFLUAR 20X/1.0 corr nd = 1.45 objective. The correcting ring was adjusted at 1.47.
3.For samples cleared with CUBIC, the microscope imaging chamber was filled with 20mL of Reagent-2 (refractive index ≈ 1.48) and acquisitions were done using LSFM 10X illumination objectives and Clr Plan–NEOFLUAR 20X/1.0 corr nd = 1.45 objective. The correcting ring was adjusted at 1.48.
Comparison of intestinal organoids transparency after different clearing methods
Fluorescence preservation after different clearing methods on intestinal organoids
Comparison of different acquisition modes
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