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

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

2. 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

3. 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

4. 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.

5. 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

6. 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

7. 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?)

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

9. Practice In “Results” section B lymphocytes reduced EV71 infection. B lymphocytes were detected in the brain of EV71-infected patients by immunohistochemical staining. EV71 infection increased the number of B lymphocytes in the brain of infected mice when assayed by flow cytometry. Increased virus titers and higher mortality rate were observed in B-lymphocyte deficient mice infected with EV71. CD4 T lymphocytes reduced EV71 infection. CD4+ T lymphocytes were detected in the brain of EV71-infected patients by immunohistochemical staining. EV71 infection increased the number of CD4+ T lymphocytes in the brain of infected mice when assayed by flow cytometry. Increased virus titers and higher mortality rate were observed in CD4+ T-lymphocyte deficient mice infected with EV71. CD8 T lymphocytes reduced EV71 infection. CD8+ T lymphocytes were detected in the brain of EV71-infected patients by immunohistochemical staining. EV71 infection increased the number of CD8+ T lymphocytes in the brain of infected mice when assayed by flow cytometry. Increased virus titers and higher mortality rate were observed in CD8+ T-lymphocyte deficient mice infected with EV71.

10. Immunohistochemical staining The brain stem specimens of EV71-infected patients were obtained from a 9-month-old girl with consent from patient’s family. The presence of B, CD4+ T, and CD8+ lymphocytes in the brain specimens was detected by CD19, CD4, and CD8 antibodies (Sigma) as previously described (1). Cells and virus Vero cells were maintained in RPMI medium containing 10% fetal bovine serum according to American Type Culture Collection instructions. EV71 strain 4643 was propagated and titrated in Vero cells as previously described (18). (Why cells were described before virus? ) Infection of mice Wild type C57BL/6, B-lymphocyte (Igh-6tm1Cgn/J), CD4+ T-lymphocyte (Cd4tm1Mak/J), and CD8 + T-lymphocyte (Cd8tm1Mak/J), deficient mice were purchased from The Jackson Laboratory. Mice were infection with 1 x 107 plaque forming units (PFU) of EV71 strain 4643 by oral inoculation as previously described (18). The survival of infected mice were monitored for 2 weeks. The brain of infected mice were harvested to determine viral load by plaque assay on Vero cells. Flow cytometry

11. Use of human subjects or specimens or animals for studies: For example: Human DV3 immune serum was obtained with consent from an infected patient. For example: All animal experiment protocols were approved by the Laboratory Animal Committee at National Cheng Kung University.

12. You should acknowledge reagents kindly provided by other researchers in the “MMs” or Acknowledgements. • In the “MMs” : • Plasmid A (kindly provided by G. -C. Perng) was used to construct virus. • B mice (kindly provided by D. Coen in Harvard Medical School) were • used for experiments. • In the Acknowledgments: • We thank G. -C. Perng for providing plasmid A and D. Coen for providing mice B.

13. Consistence in citing sources (companies) based on the format of the journal you choose The Fas expression on infected cells was detected by anti-Fas antibody (Endogen, Woburn, MA). Glycerol used in the study was purchased from Endogen (Woburn, Mass.). Mouse specific monocyte antibody was obtained from PharMingen. IL-6 production was measured by a ELISA kit (Sigma, Saint Louis, MO). When do you need to cite a source for a reagent? Does a common, non-specific reagent, like glycerol and ethanol, need a source?

14. Homework: • Read the “Results” and analyze the “MMs” of your paper. • Practice to write your own “MMs” • Do not forget to address human subjects or animal studies. • Please cite companies based on the format of your journal.