Transcript Materials and Methods

What is “Materials and Methods” and what should be in this section?
Materials and Methods. The Materials and Methods
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.
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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.
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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.
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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).
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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 described previously (40).
Flow cytometry. Infected B cells were washed with phosphatebuffered 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.
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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 logrank test. All P values are for two-tailed significance tests.
Subheadings
Previous papers in the laboratory are good examples.
Chronological order (Which should come first?)
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Match the sequence of the “Materials and Methods” with that
of the “Results”.
However, related methods should be described together.
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 deficiency 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 deficiency 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 deficiency mice infected with EV71.
Immunohistochemical staining
The brain stem specimens of EV71-infected patients were obtained from a 9month-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), deficiency 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
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.
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You should acknowledge reagents kindly provided by other
researchers in the Materials and Methods or in the
Acknowledgement.
In the Materials and Methods:
•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 Acknowledgment:
We thank G. -C. Perng for providing plasmid A and D. Coen for providing mice B.
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Consistence in writing
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 does the source of a reagent is needed?
Does a common, non-specific reagent, like glycerol, need a source?
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