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

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).

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