T-cell dependent response (TDAR)
To assess the role of the PPAR in the humoral response to a T-cell-dependent antigen, B6.129S4-Ppartm1GonzN12 (PPAR KO) and background-matched wild-type (WT) C57BL/6-Tac female mice were purchased from Taconic Farms (Rensselaer, NY). The vendor website (http://www.taconic.com/cs/Satellite?

blobcol¼urldocument&blobkey¼id&blobtable=TA_Document &blobwhere¼1347208841885&ssbinary¼true) includes a detailed description of the KO strain. 
All animals were delivered to animal facilities at the USEPA that are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). All animals were 6–7-weeks-of-age at delivery. Once at the USEPA, the animals were housed in poly-carbonate cages with hardwood chip bedding (Beta Chip) in groups of six (PPAR KO andWT) or four (C57BL/6N) and allowed to acclimate for at least 10 days before dosing began. They were provided a 12-h light:dark cycle (light, 06:00–18:00 hours; dark, 18:00–06:00 hours), maintained at 22.3 [± 1.1]C and 40–60% relative humidity, and given ad libitum access to both food (5P00 Prolab RMH 3000, PMI Nutrition International, Richmond, IN) and water. The Institutional Animal Care and Use Committee of the National Health and Environmental Effects Research Laboratory, US EPA, approved all procedures in advance. 

T-cell independent response (TIAR)
To evaluate immunophenotype in animals immunized with a T-cell-dependent antigen, C57BL/6N female mice were purchased from Charles River Laboratories (Raleigh, NC), the same source and strain of mouse used in our previous work (DeWitt et al., 2008).To evaluate T- or B-cell targeting by PFOA, C57BL/6N female mice were purchased from Charles River Laboratories and delivered to the East Carolina University (ECU) Brody School of Medicine (BSOM) animal facility (accredited by AAALAC). All animals were 6–7 weeks-of-age at delivery. Once at the ECU animal facility, animals were housed in  polycarbonate cages with corncob bedding (Bed O’Cobs) in groups of eight and allowed to acclimate for at least 10 days before dosing began. The mice were provided a 12-h light:dark cycle (light, 06:00–18:00 hours; dark, 18:00–06:00 hours), maintained at 22.8 [± 3] C and 30–70% relative humidity, and given ad libitum access to both food (5P00 Prolab RMH 3000) and water. 

10 days; steady state animals not immunized
Four C57BL/6N mice from each dose were euthanized after 10,13, or 15 days of dosing and spleens were harvested. Animals euthanized after 10 days of exposure were used to determine the effects of PFOA exposure at immunological steady state; i.e. in animals that were exposed to PFOA but not immunized. The remaining mice were immunized with SRBC on Day 11 to stimulate a primary immune response and euthanized on Days 13 and 15 to assess the effects of immunization on lymphocyte subset distributions over time and to determine if PFOA exposure had an impact on sub-sets. Spleens from the mice were rapidly removed after euthanasia and immediately placed into 4ml cold Hanks Balanced Salt Solution (HBSS without CaCl2, MgSO4, or phenol red) and processed into single cell suspensions using a Stomacher 80 blender. Red blood cells were lysed with 0.84% [w/v] ammonium chloride, cells were washed twice with HBSS, and then resuspended in cold PBS containing 1% BSA with 0.1% sodium azide (flow cytometry staining buffer). The total number of nucleated lymphocytes was determined on a Beckman Coulter Counter (Beckman Coulter, Indianapolis, IN) and cell viability was determined by trypan blue exclusion. Cells were adjusted to 107/ml in flow cytometry staining buffer and kept on ice until immunofluorescent labeling.
Single-cell suspensions were pre-incubated for 10 min on ice with Fc-receptor blocking antibody to reduce non-specific binding of immunoglobulins, followed by incubation on ice for 30 min with the appropriate concentration of monoclonal antibody diluted in staining buffer. The following monoclonal antibodies were used:Purified rat anti-mouse CD16/CD32 (Fc block), fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4, phycoerythrin (PE)-anti-mouse NK1.1, PE-Cy7-anti-mouse CD8a, allophycocyanin (APC)-anti-mouse CD3", APC-Cy7-anti-mouse CD45R. Isotype controls were prepared by labeling cells at the same protein concentration as the test antibody. Isotype control antibodies included: FITC rat IgG2a, APC-Cy7 rat IgG2a, R-PE mouse IgG2a, APC hamster IgG1, and PE-Cy7 rat IgG2a. All antibodies were purchased from BD Biosciences (San Jose, CA); PE-Cy7 Rat IgG2a was from eBioscience (San Diego, CA). After three washes, cells were fixed with 0.05% formaldehyde in PBS prior to analysis.