1. Preparation steps
<ol>
	<li>Prepare Muller-Hinton broth (MHB) by adding 25 g of MH broth to 1 L of distilled water and mix. Autoclave at 121 &#176;C for 2.5 h. Then, store the autoclaved media at room temperature or in the fridge.</li>
	<li>Subculture the bacteria in question (E. coli ESBL) on the agar media using the four-quadrant streaking method and incubate overnight at 37 &#176;C.
	<ol>
		<li>Using a sterile loop, take one colony and spread it in the first half of the MacConkey agar plate by doing close parallel streaks.</li>
		<li>Using the loop, spread the bacteria in the second quadrant by streaking it from the first quadrant.</li>
		<li>From the second quadrant, extend the streaks in the third quadrant using close parallel streaks.</li>
		<li>Spread the bacteria in the center of the fourth quadrant by streaking it from the third quadrant.</li>
	</ol>
	</li>
</ol>
2. Panel preparation
<ol>
	<li>Place four 96-well plates next to each other to form a square. Using a tape, tape their bottom together.</li>
	<li>Repeat this step to obtain four panels each containing 4 plates and name them A1, A2, A3, and A4.</li>
	<li>Add 50 &#181;L of MH broth to the wells between column 2 and column 11 in the 16 96-well plates of the four panels.</li>
	<li>Add 200 &#181;L of MH broth to the well H12 serving as the negative control well in the 16 96-well plates of the four panels.</li>
	<li>Add 150 &#181;L of MH broth to the wells A1 and H1 serving as positive control wells in the 16 96-well plates of the four panels.</li>
</ol>
3. Drug 1, Cefotaxime, serial dilution
<ol>
	<li>To a conical tube, add 15 mL of sterile dH2O.
	<ol>
		<li>Calculate the volume to be removed from the stock solution of the drug following the formula C1V1 = C2V2. Thus, V1 = (C2 x 15 mL) / C1. 
		NOTE: In our case, the Cefotaxime stock solution is 105 &#956;g/mL and C2 is 256 &#956;g/mL; thus, V1 = 38.4 &#181;L.</li>
	</ol>
	</li>
	<li>Remove the calculated volume from the 15 mL of sterile dH2O, and then add the drug.</li>
	<li>Pipette 50 &#181;L of the prepared drug solution into each well in column 11 and column 12 except H12.</li>
	<li>Start the serial dilution by removing 50 &#181;L from column 11 and putting it into the corresponding wells in column 10, and then from 10 until reaching column 2 where the 50 &#181;L taken from column 2 will be discarded.</li>
	<li>Repeat steps 3.4 and 3.5 for all the 16 96-well plates of the four panels.</li>
</ol>
4. Drug 2, Amkacin, serial dilution
<ol>
	<li>To a conical tube, add 10 mL of sterile dH2O.</li>
	<li>Calculate the volume to be removed from the stock solution of the drug following the formula C1V1 = C2V2. Thus, V1 = (C2 x 10 mL) / C1. 
	NOTE: In our case, the Amikacin stock solution is 103 &#956;g/mL and C2 is 64 &#956;g/mL; thus, V1 = 64 &#181;L.</li>
	<li>Remove the calculated volume from the 10 mL of sterile dH2O, and then add the drug.</li>
	<li>Take eight separate 96-well plates.</li>
	<li>To each plate, add 100 &#181;L of MHB to the wells between rows G and B.</li>
	<li>Add 100 &#181;L of the previously prepared drug 2 solution to the wells of row G.</li>
	<li>Dilute serially from row G to row B by taking 100 &#181;L from each well and finally discard the 100 &#181;L from the wells of row B.</li>
	<li>Repeat steps 4.5, 4.6, and 4.7 to prepare eight plates.</li>
</ol>
5. Transfer of drug 2 to the four panels
<ol>
	<li>Pipette 50 &#181;L of drug 2 from the wells between rows G and B into the corresponding wells in each plate in the four panels. One prepared 96-well plate contains 100 &#181;L of drug 2 that is enough for two plates in one panel.</li>
</ol>
6. Drug 3, Levofloxacin, addition
<ol>
	<li>To four different conical tubes, add 14 mL of sterile dH2O.</li>
	<li>Calculate the volume to be removed from the stock solution of the drug following the formula C1V1 = C2V2. Thus, V1 = (C2 x 14 mL) / C1. 
	NOTE: In our case, Levofloxacin is prepared in four different concentrations from a stock solution of 5 x 103 &#956;g/mL. 
	C1 = 2 &#956;g/mL, V1 = 5.6 &#181;L 
	C2 = 4 &#956;g/mL, V1 = 11.2 &#181;L 
	C3 = 8 &#956;g/mL, V1 = 22.4 &#181;L 
	C4 = 16 &#956;g/mL, V1 = 44.8 &#181;L</li>
	<li>Remove the calculated volume from the 14 mL of sterile dH2O from each tube, and then add the drug.</li>
	<li>After preparing the required concentrations of the third drug, take 50 &#181;L and add it to the corresponding wells between rows B and G and columns 2 and 12 in the corresponding plate in each panel where C1 corresponds to the four P1 plates in the four panels, C2 corresponds to the four P2 plates in the four panels, C3 corresponds to the four P3 plates in the four panels, and C4 corresponds to the four P4 plates in the four panels.</li>
</ol>
7. Drug 4, Trimethoprim-sulfamethoxazole, addition
<ol>
	<li>To a conical tube, add 14 mL of sterile dH2O.</li>
	<li>Calculate the volume to be removed from the stock solution of the drug following the formula C1V1 = C2V2. Thus, V1 = (C2 x 14 mL) / C1. 
	NOTE: In our case, trimethoprim-sulfamethoxazole is prepared in four different concentrations from a stock solution of 48 x 103 &#956;g/mL. 
	C1 = 512 &#956;g/mL, V1 = 149.33 &#181;L 
	C2 = 1024 &#956;g/mL, V1 = 298.66 &#181;L 
	C3 = 2048 &#956;g/mL, V1 = 597.33 &#181;L 
	C4 = 4096 &#956;g/mL, V1 = 1 194.66 &#181;L</li>
	<li>Remove the calculated volume from the 14 mL of sterile dH2O of each tube, and then add the drug.</li>
	<li>After preparing the required concentrations of the fourth drug, take 50 &#181;L and add it to the corresponding wells between rows B and G and columns 2 and 12 in the four plates in the corresponding panel where C1 corresponds to panel 1, C2 corresponds to panel 2, C3 corresponds to panel 3, and C4 corresponds to panel 4.</li>
</ol>
8. Preparation and addition of bacterial inoculum E. coli ESBL
<ol>
	<li>Using a sterile loop, transfer one colony of the bacterial isolate E. coli ESBL previously cultured on a plate into 2 mL of sterile MHB and vortex.</li>
	<li>Check for the turbidity where it should be 0.5 McFarland using a densitometer.</li>
	<li>Add 80 mL of sterile MH broth to a sterile urine cup.</li>
	<li>Add bacterial inoculum from the 0.5 McFarland inoculum to the urine cup following C1V1 = C2V2, where V1 = (106 x 80 mL) / 108 = 800 &#181;L.</li>
	<li>Pipette 50 &#181;L of the inoculum solution 106 CFU/mL into each well except H12, which is the sterility control well.</li>
	<li>Incubate the panels at 37 &#176;C overnight.</li>
	<li>After incubation, add 50 &#181;L of Iodotetrazolium to record the growth in the wells.</li>
</ol>
9. Protocol for the FIC template (Supplemental File)
<ol>
	<li>Write the highest concentration of drug 1 (Cefotaxime) in the yellow cell in panel A.</li>
	<li>Write the highest concentration of drug 2 (Amikacin) in the yellow cell in panel B.</li>
	<li>Write the highest concentration of drug 3 (Levofloxacin) in the yellow cell in panel C.</li>
	<li>Write the highest concentration of drug 4 (Trimethoprim-sulfamethoxazole) in panel D.</li>
	<li>Trace the Redline (Growth/no Growth interface) by highlighting the wells having growth in red.</li>
	<li>Write the MIC of drug 1 in the table of drug 1 (ATB1).</li>
	<li>Write the MIC of drug 2 in the table of drug 2 (ATB2).</li>
	<li>Write the MIC of drug 3 in the table of drug 3 (ATB3).</li>
	<li>Write the MIC of drug 4 in the table of drug 4 (ATB4).</li>
	<li>To calculate FIC for ATB1
	<ol>
		<li>Determine the wells on the Growth/no Growth interface.</li>
		<li>In table ATB1, double click on the cell next to FIC1 and drag the yellow cell on the left of panel A to the first selected well.</li>
		<li>Repeat step 9.10.2 for every selected well where well 1 corresponds to FIC1, well 2 corresponds to FIC2, and so on.</li>
	</ol>
	</li>
	<li>To calculate the FIC for ATB2
	<ol>
		<li>In table ATB2, double click on the cell next to FIC1 and drag the yellow cell on the left of panel B to the first pre-selected well.</li>
		<li>Repeat step 9.11.1 for every pre-selected well.</li>
	</ol>
	</li>
	<li>To calculate the FIC for ATB3
	<ol>
		<li>In table ATB3, double click on the cell next to FIC1 and drag the yellow cell on the left of panel C to the first pre-selected well.</li>
		<li>Repeat step 9.12.1 for every pre-selected well.</li>
	</ol>
	</li>
	<li>To calculate the FIC for ATB4
	<ol>
		<li>In table ATB4, double click on the cell next to FIC1 and drag the yellow cell on the left of panel D to the first pre-selected well.</li>
		<li>Repeat step 9.13.1 for every pre-selected well.</li>
	</ol>
	</li>
	<li>In the table labeled ATB1+2+3+4, sum the FIC1 of each ATB automatically. The same will occur for the other FICs (FIC2, FIC3, and so on).
	<ol>
		<li>In the cell containing FIC and highlighted in yellow, double click on it and select the summed &#931;FICs from table ATB1+2+3+4.</li>
	</ol>
	</li>
	<li>Repeat these steps for each of the nine sheets representing the nine plates. 
	&#8203;NOTE: In the sheet FIC all, the table will show the final summed FIC with the interpretation of the obtained value.</li>
</ol>
10. MIC determination using the microdilution assay for the four drugs
<ol>
	<li>Label four different rows with the abbreviation of each tested drug. For example, CTX for Cefotaxime, AMK for Amikacin, LEVO for Levofloxacin, and SXT for Trimethoprim-sulfamethoxazole.</li>
	<li>Pipette 200 &#181;L of sterile Muller Hinton broth into well number 1 and well number 12 in each used row. Well number 12 will serve as the negative control well.</li>
	<li>Pipette 100 &#181;L of sterile Muller Hinton broth to the wells 2 till 11 in each used row.</li>
	<li>Calculate the volume of each drug to be added using the formula C1V1 = C2V2. Thus, V1 = (C2 x 4 x V2) / C1. V2 is the final volume in the wells which is 200 &#181;L, C1 is the concentration of the stock solution, and C2 is the initial concentration that we need to have in the first well. 
	NOTE: Here, we use the following: 
	Cefotaxime: C1 = 105 &#956;g/mL, where we dilute it to 104 &#956;g/mL, C2 = 256 &#956;g/mL; thus, V1 = 20.48 &#181;L 
	Amikacin: C1 = 103 &#956;g/mL, C2 = 64 &#956;g/mL; thus, V1 = 51.2 &#181;L 
	Levofloxacin: C1 = 5 x 103 &#956;g/mL, where we dilute it to 5 x 102 &#956;g/mL, C2 = 16 &#956;g/mL; thus, V1 = 25.6 &#181;L 
	Trimethoprim-Sulfamethoxazole: C1 = 48 x 103 &#956;g/mL, C2 = 4096 &#956;g/mL; thus, V1 = 68.26 &#181;L</li>
	<li>Pipette the required volume for each drug in the first well of the corresponding row after removing the same volume from the 200 &#181;L broth to obtain a total volume of 200 &#181;L after the addition of the drug.</li>
	<li>Serially dilute by removing 100 &#181;L from well 1 into well 2 and so on until reaching well 10 where the 100 &#181;L removed from well 10 will be discarded. Note that well 11 serves as the positive control well.</li>
	<li>Pipette 100 &#181;L of 106 prepared bacterial inoculum into each well in each used row except for well number 12 serving as the negative control.</li>
	<li>Incubate at 37 &#176;C overnight.</li>
</ol>

<ol>
	<li>Ibezim, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+resistance+to+antibiotics.">Microbial resistance to antibiotics.</a> African Journal of Biotechnology. 4, 1606-1611 (2006).</li><li>Ventola, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibiotic+resistance+crisis:+part+1:+causes+and+threats.">The antibiotic resistance crisis: part 1: causes and threats.</a> P &amp; T: A Peer-Reviewed Journal for Formulary Management. 40 (4), 277-283 (2015).</li><li>Alanis, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+antibiotics:+Are+we+in+the+post-antibiotic+era.">Resistance to antibiotics: Are we in the post-antibiotic era.</a> Archives of Medical Research. 36 (6), 697-705 (2005).</li><li>Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotics+at+the+crossroads.">Antibiotics at the crossroads.</a> Nature. 431 (7011), 899-902 (2004).</li><li>Bollenbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+interactions:+mechanisms+and+implications+for+drug+discovery+and+resistance+evolution.">Antimicrobial interactions: mechanisms and implications for drug discovery and resistance evolution.</a> Current Opinion in Microbiology. 27, 1-9 (2015).</li><li>Mehta, K. C., Dargad, R. R., Borade, D. M., Swami, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+antibiotic+resistance+in+common+infectious+diseases:+role+of+antibiotic+combination+therapy.">Burden of antibiotic resistance in common infectious diseases: role of antibiotic combination therapy.</a> Journal of Clinical and Diagnostic Research: JCDR. 8 (6), (2014).</li><li>Chanda, S., Rakholiya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+therapy:+Synergism+between+natural+plant+extracts+and+antibiotics+against+infectious+diseases.">Combination therapy: Synergism between natural plant extracts and antibiotics against infectious diseases.</a> Science against Microbial Pathogens: Communicating Current Research and Technological Advances. , (2011).</li><li>Cottarel, G., Wierzbowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+drugs,+an+emerging+option+for+antibacterial+therapy.">Combination drugs, an emerging option for antibacterial therapy.</a> Trends in Biotechnology. 25 (12), 547-555 (2007).</li><li>Kristiansen, J., Amaral, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+management+of+resistant+infection+with+non-antibiotics.">The potential management of resistant infection with non-antibiotics.</a> The Journal of Antimicrobial Chemotherapy. 40, 319-327 (1997).</li><li>Tamma, P. D., Cosgrove, S. E., Maragakis, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+therapy+for+treatment+of+infections+with+gram-negative+bacteria.">Combination therapy for treatment of infections with gram-negative bacteria.</a> Clinical Microbiology Reviews. 25 (3), 450-470 (2012).</li><li>Leekha, S., Terrell, C. L., Edson, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+principles+of+antimicrobial+therapy.">General principles of antimicrobial therapy.</a> Mayo Clinic Proceedings. 86 (2), 156-167 (2011).</li><li>Eliopoulos, G. M., Eliopoulos, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+combinations:+Should+they+be+tested.">Antibiotic combinations: Should they be tested.</a> Clinical Microbiology Reviews. 1 (2), 139-156 (1988).</li><li>Doern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+does+2+plus+2+equal+5?+A+review+of+antimicrobial+synergy+testing.">When does 2 plus 2 equal 5? A review of antimicrobial synergy testing.</a> Journal of Clinical Microbiology. 52 (12), 4124-4128 (2014).</li><li>Stein, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+dimensional+checkerboard+synergy+analysis+of+colistin,+meropenem,+tigecycline+against+multidrug-resistant+clinical+klebsiella+pneumonia+isolates.">Three dimensional checkerboard synergy analysis of colistin, meropenem, tigecycline against multidrug-resistant clinical klebsiella pneumonia isolates.</a> PloS One. 10 (6), 0126479 (2015).</li><li>Langeveld, W. T., Veldhuizen, E. J. A., Burt, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+between+essential+oil+components+and+antibiotics:+a+review.">Synergy between essential oil components and antibiotics: a review.</a> Critical Reviews in Microbiology. 40 (1), 76-94 (2014).</li><li>Pankey, G., Ashcraft, D., Kahn, H., Ismail, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-kill+assay+and+Etest+evaluation+for+synergy+with+polymyxin+B+and+fluconazole+against+Candida+glabrata.">Time-kill assay and Etest evaluation for synergy with polymyxin B and fluconazole against Candida glabrata.</a> Antimicrobial Agents and Chemotherapy. 58 (10), 5795-5800 (2014).</li><li>Odds, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy,+antagonism,+and+what+the+chequerboard+puts+between+them.">Synergy, antagonism, and what the chequerboard puts between them.</a> The Journal of Antimicrobial Chemotherapy. 52 (1), 1 (2003).</li></ol>

The Quadruple&#160;Checkerboard method resembles the checkerboard and the three dimensional checkerboard in its protocol. However, certain crucial steps should be taken into consideration to avoid errors during the experiment.
Make sure to test for the MIC of each drug against the tested isolate before starting the protocol to know what are the concentrations that are needed to start the dilutions with for drug 1 and drug 2 that need to be serially diluted in the plates. Concerning drug 3 and ...

With the increase in resistance due to the overuse and misuse of antibiotics1,2, the need to develop new drugs and agents to treat bacterial infections has become crucial. New approaches such as developing new drugs are very important to overcome the resistance crisis. However, the pharmaceutical industry is not interested in developing new antimicrobial agents. Moreover, if new drugs are developed, bacteria will keep on evolving and developing resistance against these new drugs3,4. Thus, the problem of resistance will not be solved, making the need for another approach a must that should be considered and studied to overcome bacterial resistance.
Drug combination is a very important concept for treating bacterial infections mainly those that are caused by multidrug resistant pathogens5,6. It decreases the course of treatment, decreases the dose given; thus, decreasing the toxicity of the given drug, helps in decreasing the rate of resistance development and, in a way, sensitizes the bacteria to the given drugs as described in the concept of collateral sensitivity5,7,8,9.
Resistance development to one drug requires a single mutation; however, resistance development to a combination of drugs targeting multiple pathways requires several independent mutations that are slowed down by this combination. An example to decreased resistance while using combination therapy is the decreased rate of resistance to Rifampin in Mycobacterium Tuberculosis10. Another example is a study done by Gribble et al. that showed the rate of emergence of resistant strains in patients taking Piperacillin alone to be higher than in those taking a combination of carboxypenicillin and aminoglycoside10. Studies have shown that resistance development to aminoglycosides in evolving bacteria made these strains sensitive to various other drugs5. The combination between the beta-lactam class drug amoxicillin and the lactamase inhibitor clavulanic acid showed success in treating resistant bacterial strains8.
Decreasing the time of treatment is a good advantage resulting from drug combinations. For example, a therapy of combined penicillin or ceftriaxone with gentamicin for 2 weeks will give the same efficacy given by penicillin or ceftriaxone alone when given for 4 weeks11. Combining drugs allows for the usage of lower dosages of drugs that are not effective when given alone such as the Sub-MICs. The example of sulfonamides can be given where the use of triple-sulfonamides minimizes, at lower doses, the toxicity produced which is crystal formation or crystalluria when using insoluble sulfonamides at full doses12.
Thus, decreasing the dosage given and the time of treatment will eventually decrease the toxicity of the drugs on the body. The idea of developing methods to assess the interaction between combined drugs is very important. In one study, the results showed that combination therapy is more effective for the treatment of resistant species of Acinetobacter and P. aeruginosa8.
Giving drugs in combination 
There are different methods by which we can study drug combinations, such as the checkerboard method, the time-kill curve method, and the E-test method13. The checkerboard method can study all the possible combinations between the two drugs in question in one experiment itself. In addition, it was developed to study a combination of three drugs14. Now, we extend this to study a combination of four drugs mainly for the treatment of multidrug-resistant pathogens.
The time-kill curve assay is usually performed to test for the bactericidal effect of a certain drug. It was also used to test for the effect of drug combinations where several drugs are combined at specific concentrations. This protocol requires the preparation of several sterile tubes or cups where in each cup we add the broth, combination of drugs, and the required bacterial strain. After incubation and recording of the optical density at several time points, the results are compared with the normal growth rate of the used strain to see whether the growth rate increased, decreased, or did not change13.
E-test method is usually done to test for the minimal inhibitory concentration (MIC) where a strip containing a gradient concentration of the drug in question is put on an inoculated plate. It was also used to test the combination between two drugs where two strips are added to the plate in a perpendicular manner intersecting at their MICs13.
According to literature, there is no gold standard to define and study synergy; thus, it is difficult to assess which one of the methods used to study combination is better and which one produces better and more reliable results mainly13. However, Time-kill assay is labor intensive, time consuming, and expensive15,16, while the E-test method is developed to study a combination between two drugs only. Checkerboard can study all the possible combinations between the two drugs tested and this is why this technique has been chosen to be developed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1000 &#181;L tips</td>
			<td>Citotest</td>
			<td>4330000402</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L tips</td>
			<td>Citotest</td>
			<td>4330-0013-17</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>corning</td>
			<td>430828</td>
			<td>For drug 3 and 4 preparation</td>
		</tr>
		<tr>
			<td>5 mL polysterene round-bottom Tube</td>
			<td>Falcon</td>
			<td>352058</td>
			<td>For 0.5 MacFarland bacterial inoculum preparation</td>
		</tr>
		<tr>
			<td>90mm petri dishes</td>
			<td>JRZ Plastilab</td>
			<td></td>
			<td>As bed for the solutions to be added using the multichannel pipette</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>corning</td>
			<td>3596</td>
			<td>For serial diltuion and combining drugs</td>
		</tr>
		<tr>
			<td>Bactrim 200, 40 mg (Trimethoprim-sulamethoxazole</td>
			<td>By CRNEXI SAS Fontenay-sous-Bois, France</td>
			<td>10177403</td>
			<td>Drug 4</td>
		</tr>
		<tr>
			<td>Ceforane, 1 g (Cefotaxime)</td>
			<td>PHARCO Pharmaceuticals</td>
			<td>24750/2006</td>
			<td>Drug 1</td>
		</tr>
		<tr>
			<td>Densitometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. Coli ESBL strain</td>
			<td>Retreived as a medical strain from the Saint-George Hospital Lebanon</td>
			<td></td>
			<td>Bacterial strain</td>
		</tr>
		<tr>
			<td>Mac Conkey + crystal violet agar</td>
			<td>BIO-RAD</td>
			<td>64169508</td>
			<td>For making agar plates used for subculturing</td>
		</tr>
		<tr>
			<td>Miacin 500 mg/2 mL (Amikacin)</td>
			<td>HIKMA Pharmaceuticals</td>
			<td>2BXMIA56N-AEF</td>
			<td>Drug 2</td>
		</tr>
		<tr>
			<td>Muller-Hinton Broth</td>
			<td>BIO-RAD</td>
			<td>69444</td>
			<td>For making bacterial media</td>
		</tr>
		<tr>
			<td>Multichannel Pipette</td>
			<td>Thermo Scientific</td>
			<td>GJ54761</td>
			<td>For serial dilution and addition of media, bacteria and drugs</td>
		</tr>
		<tr>
			<td>Paper Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel pipettes</td>
			<td>Thermo Scientific</td>
			<td>OH19855 HH40868</td>
			<td>For the addition of media, bacteria and drugs</td>
		</tr>
		<tr>
			<td>Tavanic, 500 mg (Levofloxacin)</td>
			<td>sanofi aventis</td>
			<td>221937/2009</td>
			<td>Drug 3</td>
		</tr>
	</tbody>
</table>

quadruple-checkerboard: a modification of the three-dimensional checkerboard for studying drug combinations

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The Quadruple&#160;checkerboard, also called the Q-checkerboard, aims at maximizing the number of possible combinations that can be obtained between four drugs in one experiment to minimize the time and work needed to accomplish the same results with other protocols. This protocol is based on the simple micro dilution technique where the drugs are diluted and combined together in several 96-well plates.
In the first set of 96-well plates, Muller-Hinton broth is added followed by the first required drug (e.g., Cefotaxime here) to serially dilute it. After the first step is done, another set of 96-well plates is used to dilute the second drug (e.g., Amikaci), which will be transferred by removing a specific volume of drug 2 and put in the corresponding wells in the first set of 96-well plates that contains drug one. The third step is done by adding the required concentrations of the third drug (e.g., Levofloxacin), to the appropriate plates in the initial set containing combination of drug 1 and 2. The fourth step is done by adding the required concentrations of the fourth drug (e.g., Trimethoprim-sulfamethoxazol) into the appropriate plates in the first set. Then, E. coli ESBL bacterial inoculum will be prepared and added.
This method is important to evaluate all the possible combinations and has a wider range of possibilities to be tested furthermore for in vivo testing. Despite being a tiring technique requiring a lot of focus, the results are remarkable and time saving where a lot of combinations can be tested in a single experiment.

Figure 2A represents the results obtained by combining Cefotaxime and Amikacin with specific concentrations of Levofloxacin and Trimethoprim-sulfamethoxazole. We can see in the left part of the figure the four plates that are schematically presented with the concentrations of the drugs in the right part of the figure. The arrows represent the wells on the Growth/no Growth interface. The colored wells are the wells that contain growth. We notice that the fourth plate does not contain growth i...

Watch this Scientific Journal Video about Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations at JoVE.com

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

This protocol describes how to study all possible combinations that can be obtained between four drugs in one single experiment. This method is based on the standard 96-well plate micro dilution assay and the calculation of fractional inhibitory concentrations (FICs) to evaluate the results.

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The ...

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4.3K Views.  University of Balamand. This protocol describes how to study all possible combinations that can be obtained between four drugs in one single experiment. This method is based on the standard 96-well plate micro dilution assay and the calculation of fractional inhibitory concentrations (FICs) to evaluate the results.

Video: Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...