Overnight cultures of bifidobacteria (20 µl) were used to inoculate RCM supplemented with 0.5% (w/v) or 1% (w/v) porcine bile (Final volume 200 µl) in a 96 well microtiter plate. Biofilms were allowed to form for 24 h at 37 °C in anaerobic conditions and were then washed three times with deionised water to remove planktonic cells and left to dry for 1 h. The biofilms were stained with 1% crystal violet (100 µl) (Sigma-Aldrich) for 1 min and then washed three times with deionised water to remove excess crystal violet stain. crystal violet stained biofilms were then solubilised with 5% (v/v) acetic acid (100 µl) (Sigma-Aldrich) and the absorbance read at a wave length of 570 nm.
Crystal violet
Manufactured by Merck Group
Sourced in United States, Germany, China, Japan, United Kingdom, France, Sao Tome and Principe, Italy, Australia, Macao, Spain, Switzerland, Canada, Belgium, Poland, Brazil, Portugal, India, Denmark, Israel, Austria, Argentina, Sweden, Ireland, Hungary
Crystal violet is a synthetic dye commonly used in laboratory settings. It is a dark purple crystalline solid that is soluble in water and alcohol. Crystal violet has a variety of applications in the field of microbiology and histology, including as a staining agent for microscopy and in the gram staining technique.
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Lab products found in correlation
Matrigel, by BD (313 mentions)
Paraformaldehyde (pfa), by Merck Group (213 mentions)
Methanol, by Merck Group (154 mentions)
Matrigel, by Merck Group (117 mentions)
Matrigel, by Corning (114 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (114 mentions)
Formaldehyde, by Merck Group (109 mentions)
Transwell chamber, by Corning (109 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (109 mentions)
Glutaraldehyde, by Merck Group (66 mentions)
Inverted microscope, by Olympus (61 mentions)
Transwell insert, by Corning (60 mentions)
Propidium iodide, by Merck Group (51 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (51 mentions)
Acetic acid, by Merck Group (49 mentions)
Phosphate buffered saline (pbs), by Merck Group (47 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (46 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (45 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (43 mentions)
Bovine serum albumin (bsa), by Merck Group (42 mentions)
5 440 protocols using crystal violet
1
Bifidobacterial Biofilm Formation Assay
2
Cell Viability Assay with Crystal Violet
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Cells were seeded at 1,000 cells/per well of a black coated, clear-bottomed 96-well plate (Greiner; Stonehouse, Gloucestershire, UK) and allowed to settle overnight. Medium from cell-containing plates was gently aspirated and replaced with 100 μl of drug-containing medium. For the control, cells were exposed to medium containing DMSO only. Cells were grown in the presence of the drug(s) for 96 hours in biological triplicates, under standard cell culture conditions. At 96 hours, cells were gently washed in PBS and fixed in 4% paraformaldehyde for 30 minutes. To each well 100μl of crystal violet solution (0.05% crystal violet (Sigma) in 20% ethanol) was added and incubated at room temperature for 30 minutes. Following PBS washes, the crystal violet stain was solubilized on plate shaker for 1 hour in 100μl of 1% sodium dodecyl sulphate (Sigma; Poole, Dorset, UK) solution in PBS. Absorbance was measured at 595nm on a Perkin Elmer Fusion α plate reader and raw data analysed with Microsoft Excel.
3
Cell Viability Assay with Crystal Violet
4
Quantifying Tumor Cell Cytotoxicity
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Resting or stimulated mouse CD8 T cells were co-cultured with matching tumor cells at fixed T cell: tumor ratio. Days of co-culture depends on different T cells or tumor cell lines used in each experiment, as indicated in the figure legend. After co-culture, T cells were removed and remaining tumor cells were analyzed. T cell cytotoxicity was assessed by tumor colony formation in which the remaining tumor cells were fixed and stained for 1 h using crystal violet solution containing 0.1% crystal violet (CV, Sigma) and 50% methanol (Honeywell). For quantification, the remaining crystal violet was solubilized in 10% acetic acid (Sigma). Absorbance of this solution was measured on an Infinite 200 Pro spectrophotometer (Tecan) at 595 nm.
5
Immunofluorescence Staining of MCF10A Colonies
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MCF10A colonies were rinsed with DPBS, fixed by incubating with 4% paraformaldehyde (EM grade, Electron Microscopy Sciences) for 20 minutes followed by incubation with pure ethanol for 10 minutes at room temperature. For crystal violet staining, fixed colonies were incubated in 0.05% w/v crystal violet (Sigma) in water for 20 minutes and excess crystal violet removed by washing 5× for 5 minutes with DPBS. For E-cadherin staining, fixed colonies were blocked by washing 3× for 5 minutes in IF buffer (0.1% triton x-100 (Sigma) and 2% BSA (Fisher Scientific) in PBS). Colonies were then incubated in IF buffer containing mouse anti-E-cadherin antibody (Cell Signaling) for 2 hours at room temperature and washed with IF buffer. For imaging, colonies were incubated for 1 hour in IF buffer containing Alexa-488 conjugated goat anti-mouse IgG in IF buffer (Invitrogen), washed with IF buffer and 5 μg/ml Hoechst 33342 (Invitrogen) added to colonies. Brightfield imaging of crystal violet stained whole colonies was performed on a Zeiss Axio Observer by stitching whole well 10× images according to manufacturer’s instructions (Carl Zeiss Microimaging). Immunofluorescence imaging of E-cadherin stained colonies was performed by taking representative fields from the center middle and edge of the colony.
6
Immunofluorescence Staining of MCF10A Colonies
7
Quantification of Biofilm Formation
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For the validation of the model, six coupons from the third MTP used for biofilm formation and three blank control coupons from the second MTP were used for quantification of biofilm formation based on biomass. 10 mL of a 0.1% crystal violet solution (containing 0.1 g/ 100 mL crystal violet (Merck, 101,418, Darmstadt, Germany) dissolved in one part of methanol (Biosolve, 13,687,802, CE Valkenswaard, The Netherlands), one part of isopropanol (Merck, 1.09634) and 18 parts of Phosphate Buffered Saline (Oxoid, BR0014G)) was added to each of the falcon tubes for 20 min and shaken (Fisher Bioblock Scientific, KL2 6118 CU 00246, Merelbeke, Belgium) at 350 rpm for the staining of the total biomass of the biofilm on the coupons. The excess stain was removed by placing the tubes under gently running tap water. Retained crystal violet was dissolved by adding 10 mL of 33% acetic acid (Merck, 1.00063) for 15 min at 350rmp. The absorbance was measured at 590 nm using a spectrophotometer (Jasco, V-660, Pfungstadt, Germany). OD-measurements of the blank control coupons were subtracted from the OD-measurements of the biofilm coupons.
8
Biofilm Formation Quantification Protocol
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Biofilm formation was assessed using the Calgary biofilm device (Ceri et al., 1999 ). Overnight cultures were diluted 200‐fold in LB medium ± 1 mM IPTG in the 96 wells of a microtiter plate. Each plate contained four replicas per construct, arranged in a way to minimize spatial influence on biofilm formation, and three biological replicates were analysed. Once the pegs were placed in the wells, and the plate was sealed to reduce evaporation. Biofilms were then grown for 24 h at 37°C without shaking. The quantitative analysis of the biofilm formation and planktonic survival was performed as described previously (Cornelissen et al., 2011 ). Briefly, the pegs were washed in LB, stained for 30 min with crystal violet (0.1 % crystal violet (Merck, Kenilworth, NJ, USA) in a 1:1:18 (v/v) isopropanol‐methanol‐PBS (phosphate‐buffered saline) solution and excess crystal violet was washed from the pegs. After a 30 min fixation step, crystal violet staining is removed from the pegs using a 30% cold acetic acid solution and quantified with the Multiskan RC.
9
Quantifying Biofilm Formation via Crystal Violet
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For the validation of the model, six coupons from the third MTP used for bio lm formation and three blank control coupons from the second MTP were used for quanti cation of bio lm formation based on biomass. 10 mL of a 0.1% crystal violet solution (containing 0.1 g/100 mL crystal violet (Merck, 101418, Darmstadt, Germany) dissolved in one part of methanol (Biosolve, 13687802, CE Valkenswaard, The Netherlands), one part of isopropanol (Merck, 1.09634) and 18 parts of Phosphate Buffered Saline (Oxoid, BR0014G)) was added to each of the falcon tubes for 20 min and shaken (Fisher Bioblock Scienti c, KL2 6118 CU 00246, Merelbeke, Belgium) at 350 rpm for the staining of the total biomass of the bio lm on the coupons. The excess stain was removed by placing the tubes under gently running tap water. Retained crystal violet was dissolved by adding 10 mL of 33% acetic acid (Merck, 1.00063) for 15 min at 350rmp. The absorbance was measured at 590 nm using a spectrophotometer (Jasco, V-660, Pfungstadt, Germany). OD-measurements of the blank control coupons were subtracted from the ODmeasurements of the bio lm coupons.
10
Colony Formation Assay Protocol
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For colony formation, cells (103/well) grown in 6-well plates had their media refreshed every 3 days for 10 days. Colonies, fixed in 4% paraformaldehyde for 10 min, were stained in 0.1% crystal violet (crystal violet, Sigma-Aldrich, Burlington, MA, USA).
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