Spleens were collected on PND 90, 6 months and 1 year for this analysis. They were transferred from NCTR to the current lab coded so that the tests were conducted blind to treatment (++ for binding).

Tissues were processed the day after collection. Splenocytes were isolated by mechanical disruption and made into single cell suspensions in RPMI 1640 medium supplemented with 10% BCS and penicillin/streptomycin. Cells were counted using a Beckman Coulter Counter.

Proliferation assay: Splenocytes were cultures in 96 well plates. Splenocytes were activated with LPS, PWM, or anti-CD3 and anti-CD28. Incorporation of [3H]-thymidine was measured using standard methods (+ for methods).

ELISA: Splenocytes were cultured in 48 well plates. IgM responses using activated splenocytes were characterized by ELISA. There is no indication that this was a specific kit that was used. (+ for methods)

Flow cytometry: Splenocytes were cultured in 48 well plates. Subpopulations were measured using the well-established flow-cytometry. Samples were processed the day after collection, presumably all conducted in the same day. (+ for methods).

Li, J. et al. 2018b

Risk of bias

HAWC