Source: https://bio-protocol.org/e1105
Timestamp: 2019-04-20 18:40:34+00:00

Document:
Dey, A. K., Bhagat, A. and Chowdhury, R. (2014). Assay for Adherence of Vibrio cholerae to Eukaryotic Cell Lines. Bio-protocol 4(8): e1105. DOI: 10.21769/BioProtoc.1105.
Vibrio cholerae (V. cholerae) colonizes the intestinal epithelium and secretes cholera toxin (CT), a potent enterotoxin that causes severe fluid loss characteristic of the disease cholera. V. cholerae is a non-invasive Gram-negative bacterium that adheres to intestinal cells as well as a variety of different cell types. A protocol for adherence of V. cholerae to various cell lines is described. The adhered bacteria can be used to examine expression of genes that are differentially expressed between adhered and unadhered bacteria or other purposes (Dey et al., 2013).
Note: For handling cell lines and bacterial cultures, aseptic techniques must be used and work should be carried out in a sterile laminar flow hood when practicable.
An aliquot of the chosen cell line (approx. 106 cells) stored in liquid nitrogen (see Note 1) was used to seed a 25 cm2 tissue culture flask containing 5 ml complete DMEM. The cultures were grown to 70-80% confluency (approx. 4 x 106 cells) at 37 °C in 5% CO2 humidified atmosphere. INT 407 was grown in low glucose complete DMEM, Hep-2, Hela and undifferentiated HT-29 were grown in high glucose complete DMEM. Differentiated (see Note 2) HT-29 was grown in no glucose complete DMEM at 10% CO2 humidified atmosphere.
The cultures were washed three times with 5 ml 1x phosphate buffered saline (PBS) and 4 ml low glucose incomplete DMEM was added.
A loopful of V. cholerae strain O395 preserved at -70 °C in LB medium containing 20% glycerol was streaked on LB agar plates and incubated at 37 °C for 16-18 h.
A loopful of V. cholerae culture from the plate was inoculated into 5 ml LB medium and grown with aeration (shaking at approx. 200 rpm in a rotary shaker) at 37 °C for 16-18 h. From this culture, a 1% inoculum (50 µl) was added to 5 ml LB medium and grown to the mid logarithmic phase (OD600 0.5) with shaking at 37 °C.
The V. cholerae culture was centrifuged (5,000 x g, 5 min), washed with 5 ml 1x PBS, and suspended in low glucose incomplete DMEM at a concentration of 109 colony forming units (CFU) per ml (Bacterial cells from approximately 2 ml logarithmic phase cultures in LB medium were suspended in 1 ml incomplete DMEM.).
Cell lines grown to 70-80% confluency in 25 cm2 flasks were used.
V. cholerae culture (200 µl, approximately 2 x 108 CFU) in low glucose incomplete DMEM was added to the 70-80% confluent cell lines, resultant multiplicity of infection (MOI) was about 50.
After addition of bacteria, the cell lines were incubated for 45 min at 37 °C in 5% CO2 humidified atmosphere for optimum adherence.
After 45 min, the supernatant was discarded and non-adhered bacteria were removed by washing the infected cell line 4 times with 5 ml 1x PBS and then low glucose complete DMEM (5 ml) was added and incubated for different periods of time (1-12 h).
After the desired time period, the supernatant was discarded and the infected cell line was washed 4 to 5 times with 5 ml 1x PBS.
1 ml of 1% Triton X-100 was added and incubated for 2 min at room temperature with gentle swirling when the cell line could be seen to lyse and detach from the flask. The total culture was collected in 1.5 ml micro centrifuge tubes and vigorous pipetting or vortexing was done. The Triton X-100 treatment lysed the eukaryotic cells but had no effect on the viability of the bacterial cells (see Note 3).
The bacterial cells were collected by centrifugation (5,000 x g, 5 min) at room temperature and the pellet was suspended in 1 ml 1x PBS.
From this suspension 10 μl was added in 990 μl of 0.9% saline solution and bacterial CFU was determined by serial dilution and plating on LB agar plates. Remaining adhered bacterial cells could be used for gene expression studies or other purposes.
Parallel experiments were performed with V. cholerae under identical conditions but without cell line as an unadhered control.
Cell lines were stored in liquid nitrogen with 10% DMSO as a cryoprotectant.
Differentiation of the HT29 cells was performed essentially as described by Huet et al. (1987).
Undifferentiated HT29 cells were grown to 70-80% confluency in high glucose complete DMEM in 25 cm2 flasks.
1 ml of a solution containing 0.5% trypsin and 1 mM EDTA was added, after about 5 min the detached cells were used to seed a 25 cm2 flask containing no glucose complete DMEM supplemented with 20 mM glucose and 1 mM galactose (day 0).
Then the following schedule was followed to gradually increase the concentration of galactose to 5 mM and decrease the concentration of glucose: (a) day 1, 15 mM glucose plus 1 mM galactose; (b) day 2, 10 mM glucose plus 3 mM galactose; (c) day 3, 5 mM glucose plus 4 mM galactose; (d) day 4, 5 mM galactose. The medium was changed every day over the period of 4 days. Once adapted, cells were grown for several passages in no glucose complete DMEM containing galactose (5 mM).
Alkaline phosphatase (AP) activity was estimated as a marker of HT29 differentiation (Luongo et al., 2002).
Differentiated and undifferentiated HT29 cells were released from the 70-80% confluent flask by treatment with 1 ml trypsin (0.5%) - EDTA (1 mM) solution, washed three times with 1 ml cold 1x PBS and lysed on ice with 1x cell lysis buffer.
For AP activity assay, 50 μl cell lysate was incubated with 1 ml p-nitrophenyl phosphate (1 mg/ml) in AP assay buffer for 30 min at room temperature and the reaction was stopped by adding 100 μl of 0.1 M K2HPO4.
AP activity was calculated as absorbance at 405 nm (A405) per mg of total cellular protein. A405 was measured against lysates incubated without substrate.
Total protein was estimated by Bradford method using the Bio-Rad protein assay dye reagent.
To examine if 1% Triton X-100 treatment had any effect on bacterial cells, a bacterial culture (OD600 0.5 approximately) was divided into 2 equal parts, one part was treated with Triton X-100 for 2 min, and CFU of the treated and untreated culture was determined. If CFU of the two cultures are comparable, it may be assumed that 1% Triton X-100 treatment had no effect on the viability of the bacterial cells used in the study.
This protocol is adapted from Huet et al. (1987); Luongo et al. (2002); and Dey et al. (2013).
Dey, A. K., Bhagat, A. and Chowdhury, R. (2013). Host cell contact induces expression of virulence factors and VieA, a cyclic di-GMP phosphodiesterase, in Vibrio cholerae. J Bacteriol 195: 2004-2010.
Huet, C., Sahuquillo-Merino, C., Coudrier, E. and Louvard, D. (1987). Absorptive and mucus-secreting subclones isolated from a multipotent intestinal cell line (HT-29) provide new models for cell polarity and terminal differentiation. J Cell Biol 105: 345-357.
Luongo, D., Mazzarella, G., Della, R. F., Maurano, F. and Rossi, M. (2002). Down-regulation of ERK1 and ERK2 activity during differentiation of the intestinal cell line HT-29. Mol Cell Biochem 231: 43-50.
How to cite: Dey, A. K., Bhagat, A. and Chowdhury, R. (2014). Assay for Adherence of Vibrio cholerae to Eukaryotic Cell Lines. Bio-protocol 4(8): e1105. DOI: 10.21769/BioProtoc.1105.

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