An Assay To Evaluate the Anticancer Effects of Lactobacillus Cell-Free Supernatant

Published: May 31, 2024

Abstract

Source: Lee, J., et al. Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells. J. Vis. Exp. (2020).

This video demonstrates an in vitro assay to study the anticancer effects of Lactobacillus cell-free supernatant (LCFS). Upon exposing colorectal cancer spheroids to varying doses of LCFS, the interaction with the bacterial metabolites in LCFS induces apoptosis in the cancer cells, disrupting the spheroid structure and leading to cell death in a dose-dependent manner.

Protocol

1. Bacterial cell cultures and preparation of Lactobacillus cell-free supernatant (LCFS) NOTE: Steps 1.2 – 1.9 are conducted in an anaerobic chamber. Prepare a De Man–Rogosa–Sharpe (MRS) agar plate and broth containing L-cysteine and sterilize by autoclaving. Pre-incubate the MRS agar plate in H2 anaerobic chamber maintained at 37 ˚C with 20 ppm oxygen. Thaw Lactobacillus bacte…

Representative Results

Figure 1: Schematic representation of spheroid formation and LCFS preparation. (A) Schematic representation images of the LCFS generation protocol are marked by (i-iv) (B) Schematics of the methylcellulose-mediated spheroid formation are marked by (i-iii).

Offenlegungen

The authors have nothing to disclose.

Materials

Applied Biosystems MicroAmp Optical Adhesive Film Thermo Fisher Scientific 4311971 100 covers
Axygen 2.0 mL MaxyClear Snaplock Microcentrifuge Tube, Polypropylene, Clear, Nonsterile, 500 Tubes/Pack, 10 Packs/Case Corning SCT-200-C 500 Tubes/Pack, 10 Packs/Case
BD Difco Bacto Agar BD 214010 500 g
BD Difco Lactobacilli MRS Broth BD DF0881-17-5 500 g
Corning Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1 Corning 21-040-CV 500 mL
Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap Corning 352235 25/Pack, 500/Case
Fetal Bovine Serum, certified, US origin Thermo Fisher Scientific 16000044 500 mL
Lactobacillus fermentum Korean Collection for Type Cultures KCTC 3112
L-Cysteine hydrochloride monohydrate Sigma-Aldrich C6852-25G 25 g
Methyl Cellulose (3500-5600mPa·s, 2% in Water at 20°C) TCI M0185 500 g
MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL Applied Biosystems 4346906 20 plates
Millex-GS Syringe Filter Unit, 0.22 µm, mixed cellulose esters, 33 mm, ethylene oxide sterilized Millipore SLGS033SB 250
Penicillin-Streptomycin (10,000 U/mL) Thermo Fisher Scientific 15140122 100 mL
RPMI-1640 Gibco 11875-119 500 mL
Trypsin-EDTA (0.25%), phenol red Thermo Fisher Scientific 25200056 100 mL
Materials/Equipment/Software
CO2 incubator Thermo fisher HERAcell 150i
Conical tube 15 ml SPL 50015
Conical tube 50 ml SPL 50050
Corning Costar Ultra-Low Attachment Multiple Well Plate Sigma-Aldrich CLS7007
Corning Costar Ultra-Low Attachment Multiple Well Plate Sigma-Aldrich CLS3471
Costar 50 mL Reagent Reservoirs, 5/Bag, Sterile Costar 4870
Countess Cell Counting Chamber Slides Thermofisher C10228
Countess II FL Automated Cell Counter invitrogen AMQAF1000
EnSpire Multimode Reader Perkin Elmer Enspire 2300
Eppendorf Research Plus Multi Channel Pipette, 8-channel Eppendorf 3122000051
Incubated shaker Lab companion SIF-6000R
Multi Gauge Ver. 3.0, Fujifilm Tokyo, Japan
Optical density (OD)LAMBDA UV/Vis Spectrophotometers Perkin Elmer Waltham, MA, USA
Phase-contrast microscope Olympus Tokyo, Japan
SPL microcentrifuge tube 1.5mL SPL 60015
SPL Multi Channel Reservoirs, 12-Chs, PS, Sterile SPL 21012
Vibra-Cell Ultrasonic Liquid Processors SONICS-vibra cell VC 505 500 Watt ultrasonic processor
Vinyl Anaerobic Chamber COY LAB PRODUCTS

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An Assay To Evaluate the Anticancer Effects of Lactobacillus Cell-Free Supernatant. J. Vis. Exp. (Pending Publication), e22261, doi: (2024).

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