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Immunostaining protocol of spheroid in NCP
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Introduction

Immunostain technique for cultured cells has two type methods. One of the methods is needed manipulation of sample embedding into OTC compound or paraffin, and the other one is directly conducted in the culture slide or plate.
Here, we introduce the immunostain method for spheroid cultured in NanoCulture Plate.

Materials, reagents and equipment

1. Cell
BT-474(human breast cancer cell line) ATCC Cat# HTB-20™
2. Reagents
Media RPMI1640 supplemented 10% FBS and antibiotics
(Heat-inactivated FBS must be used)
Methanol Wako Cat# 137-01823
PBS(-) Invitrogen Cat# 21600-069
Triton®X-100 nacalai tesque Cat#25987-85
BSA SIGMA Cat#A8022
Cytokeratin 7/17(C46) Antibody santa cruz biotechnology Cat# sc-8421
Alexa Fluor ®568 Invitrogen Cat# A-11004
DAPI solution Wako Cat#340-07971
3. Materials
NanoCulture Plate MS pattern,
Low-Binding, 96-well
ORGANOGENIX Cat# NCP-LS96
4. Apparatus
Fluorescent microscopy e.g.Nikon Cat# ECLIPSE TS100 (C-SHG)

Methods

【3D Cell Culture】

1. Add media (50 µL/well) in NCP
2. Centrifuge for 5 minutes at 1,000xg to remove bubbles from the edge of the well bottom.
3. Stand for 15 – 20 minutes, at room temperature or inside the CO2 incubator to remove micro-bubbles
4. Prepare HepG2 cell suspension (1×104cells/50 µL)。
5. Add HepG2 cell suspension (1×104cells/50 µL/well) in NCP (total volume of media will be 100 µL/well).Please try not to scratch well bottom which may disturb cell spheroid growth. It may results monolayer cell growing.
6. Stand for 20 – 30 minutes at ambient condition after seeding cells for better cell attachment on the plate.If NCP plate place inside the CO2 incubator before the cells attach to NCP, it may result in an uneven distribution of spheroids due to vibration and convection micro-currents while moving the plates.
7. We strongly recommend to pour autoclaved water (about 100 µL) between the well to minimize the edge effect.
8. Incubate for ~ 7 days at 5% CO2、37℃

【Immunofluorescent stain】
Note: You mustgentlyconduct the following treatments (sample-washing and buffer-changing), because the spheroids attachment to the well bottom is minimum. If spheroids are big and floating,we recommend that you collect these spheroids into collection tube by pipetting before fixation step, and then you should follow the immunostain manipulation.

9. Discard culture sup., gently.
10. Fixation: Add 100 μL chilled MeOH (-20℃) into the well and incubate at -20℃ for 10 minutes. < or Add directly 37 % formaldehyde solution of 1/10 volume of culture media into the well, and incubate at room temperature for 30 minutes >
11. Wash sample twice:Discard the fixation buffer by pipetting (do not decantation). Add 200 μL PBS (-) and incubate at room temperature for 5 minutes.
< If spheroids are lost during above treatment, you should collect these spheroids into collection tube by pipetting,and follow the immunostain manipulation. >
12. Blocking: Discard the wash buffer by pipetting (do not decantation). Add 100 μL 3% BSA / 0.1% TritonX-100 / PBS(-) into the well, and incubate at room temperature for 30 minutes.
13. Wash sample twice: Discard the Blocking buffer by pipetting (do not decantation). Add 100 μL 0.1% TritonX-100 / PBS (-) into the well, and incubate at room temperature for 5 minutes.
14. Primary antibody incubation:Add 70 μL primary antibody solution (1/100 anti-CK7/17 mouse mAb in 1 % BSA / PBS(-)) into the well and incubate at room temperature for 90 minutes. (or at 4℃ for O/N)
15. Wash sample twice: Discard the antibody solution by pipetting (do not decantation). Add 200 μL 0.1 % TritonX-100 / PBS (-) into the well, and incubate at room temperature for 5 minutes.
16. Secondary antibody incubation: Add 70 μL L secondary antibody solution (1/500 AlexaFluor®568 conjugated anti-mouse antibody in 1 % BSA / PBS (-)) and incubateat room temperature for 60 minutes.
17. Wash sample twice: Discard the antibody solution by pipetting (do not decantation). Add 200 µL 0.1 % TritonX-100 / PBS (-) into the well, and incubate at room temperature for 5 minutes.
18. Stain nuclear with 100 µL 1 µg / mL DAPI in PBS(-), and incubate at room temperature for 5 minutes.
19. Observe sample using fluorescent microscope
< You can observe samples in NCP, directly. If you do immunostain manipulation in collection tube, you should put samples on slide glass or imaging plate.>

Example of Results

【Fluorescent immunostain images of BT-474 spheroid on NCP】

  • bright field

    scale bar:100µm
  • CK7/17

  • DAPI