Scientific Reading and Writing in English Material and Methods Shu-ying Wang, Ph.D.

1. Scientific Reading and Writing in English Material and Methods Shu-ying Wang, Ph.D. Dept. Microbiology and Immunology Tel: +886-6-2353535 ext. 5634 Fax: +886-6-2082705 Email : sswang23@mail.ncku.edu.tw

2. The “Materials and Methods (MMs)” is to address “what/how you did it”.

3. Material and Methods reads like a cookbook Need to provide sufficient information so that the readers can exactlyreproduce the experiment

4. Material and methods • Subjects • Sample preparation techniques • Sample origins • Data collection protocol • Data analysis techniques • Any computer programs used • Description of equipment and its use

5. Spacing • …..previously described(30) • 2 mM, 2 h (2 hr), 2 min, 2 sec, 2 pg/ml, 1 ml, pH 6.8, • 5%, 4°C, -80°C, ~40, • 171 mm, 4 Ǻ • a=b=C=152 mm, 0.15 x 0.15 x 0.2 mm • 1-368, 22-mer DNA

6. What is “MMs” and what should be in the “MMs” section? “MMs”. The “MMs” section should include sufficient technical information to allow the experiments to be repeated. When centrifugation conditions are critical, give enough information to enable another investigator to repeat the procedure: make of centrifuge, model of rotor, temperature, time at maximum speed, and centrifugal force ( X g rather than revolutions per minute). For commonly used materials and methods (e.g., media and protein concentration determinations), a simple reference is sufficient. -continued

7. If several alternative methods are commonly used, it is helpful to identify the method briefly as well as to cite the reference. For example, it is preferable to state ‘‘cells were broken by ultrasonic treatment as previously described (9)’’ rather than to state ‘‘cells were broken as previously described (9).’’ The reader should be allowed to assess the method without constant reference to previous publications. -continued

8. Describe new methods completely and give sources of unusual chemicals, equipment, or microbial strains. When large numbers of microbial strains or mutants are used in a study, include tables identifying the sources and properties of the strains, mutants, bacteriophages, plasmids, etc. A method, strain, etc., used in only one of several experiments reported in the paper may be described in the Results section or very briefly (one or two sentences) in a table footnote or figure legend.

9. Example: Cells, viruses, and reagents. The B-cell line Raji was maintained in RPMI medium containing 10% fetal bovine serum according to American Type Culture Collection instructions. DV2 strains PL046 and M16681 were propagated in C6/36 mosquito cells and titrated on BHK cells as previously described (18). Human DV3 immune serum was obtained with consent from an infected patient. The titer of this serum was 1:12,000 against DV3 and 1:3,200 against DV2 strain PL046, as determined by measuring 50% plaque reduction in a neutralization assay using BHK cell monolayers (14). Control serum was collected from a healthy blood donor without DV-specific antibodies in serum as determined by an enzyme-linked immunosorbent assay (ELISA) as previously described (19). -continued

10. A monoclonal antibody (MAb) against the viral envelope protein was obtained from Endogen (Woburn, Mass.), and a MAb against the viral core protein with high specificity was purified from supernatant of hybridoma cells as previouslydescribed (40). Flow cytometry. Infected B cells were washed with phosphate-buffered saline (PBS) twice, fixed, permeabilized, washed, and incubated with the IgG fraction of NS1 hyperimmune serum or normal mouse serum. Cells were then washed and incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Endogen). After incubation, cells were washed twice with PBS, resuspended in PBS, and then analyzed with a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif.) as previously described. -continued

11. Cytokine measurement. Cytokines were measured by ELISA kits according to the manufacturer’s protocols. The detection limits for the cytokines (Endogen) were as follows: TNF-a, 16 pg/ml; IL-6, 11 pg/ml. • Statistical analyses. In vitro data were analyzed by Student’s t test. Viral loads in tissues were analyzed by Mann-Whitney U test. Kaplan-Meier survival curves were analyzed by log-rank test. All P values are for two-tailed significance tests. • Use subheadings to organize “MMs”. • Previous papers in the laboratory are good examples. • Chronological order of “MMs” (Which should come first?)

12. Matchthe chronological order of “MMs” with that of the “Results”. However, related methods should be described together.

13. The recombinant cED III was expressed in E. coli BL21-codonPlus(DE3)-RIL (Stratagene, La Jolla, CA). After isopropylthiogalactoside (IPTG) induction, the recombinant cED III was purified with immobilized metal affinity chromatography (IMAC) columns. The eluent from the IMAC column was then polished by using an anion exchange column (Q sepharose fast flow; GE) after dialysis against Q buffer (20 mM Tris-Cl, 1mMEDTA, pH 8.0).

14. Recombinant native FlhDC protein complex was expressed using methods described elsewhere1.Briefly, the protein was expressed in E. coli strain BL21(DE3) (B F- dcm ompT) carrying the plasmid pSW28 bearing the tandem genes of the flhDCoperon (pET24a-flhDC), and was purified by affinity chromatography (HiTrap heparin, Pharmacia) followed by size-exclusion gel-filtration (Superdex 200, Pharmacia).

15. Homework: 1. Practice to write your own “Material and Methods” section