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Ding et al. Microbiome Res Rep 2024;3:16 https://dx.doi.org/10.20517/mrr.2023.64 Page 3 of 16

[23]
selects for a gender-specific microbiota that further contributes to gender differences in immunity .

Therefore, it makes sense to assess various B. infantis strains for their ability to regulate the Th1/Th2
balance and evaluate the function in both female and male mice. This study screens the effect of eight
B. longum subsp. infantis strains on mice isolated in our laboratory previously. Its effect on the abundance
of the major innate, adaptive immune cells, the differentiation markers of Th1/Th2 were evaluated using
immunofluorescence, flow cytometry, and ELISA test, as well as the relative expression of JAK/STAT
pathway at both transcription and translation level and the gut microbiota.

METHOD AND MATERIALS
Animal experiments
The ethical number of animal study was JN.No20210915b0601125[299]. Six-week-old females and males
were specific-pathogen-free (SPF) BALB/c mice. Female (weight, 18 ± 1 g) and male (weight, 20 ± 1 g) mice
Flow cytometry and immunofluorescence analysis
(2:1) were kept in polypropylene cages at 20-26 °C and 40% to 70% humidity after one week of adaptation
with a light-to-dark cycle of 12:12 h under hygienic conditions. The mice were fed with Co-60 gamma
irradiation experimental mouse feed. Male mice were taken out after confirming that female mice were
pregnant. The pregnant mice did not receive any treatment before delivery. Both males and females were
gavaged with normal saline and different Bifidobacterium longum subsp. infantis strains I2MI
(FJSWXI2MIM1), I4MI [FJSWXI4MI (CCFM1270)], I4MNI (FJSWXI4MNIM1), I5TI (FJSWXI5TIM1),
I6TI (FJSWXI6TIM1), I8TI [FJSWXI8TI (CCFM1271)], I10TI [FJSWXI10TI (CCFM1272)], B6MNI
[BJSWXB6MNIM1 (CCFM1269)], n = 8 per group, the details of B. longum subsp. infantis can be found in
Supplementary Materials, respectively, from 1-week-old to 3-week-old with 1 × 10 CFU/day/mice
9
[Supplementary Figure 1]. B. longum subsp. infantis were adjusted to a uniform concentration after culture
and kept in a 30% sucrose solution. Additionally, the B. longum subsp. infantis groups were kept in an
identical amount of 30% sucrose solution at -80 °C, serving as the control group. The 30% sucrose solution
in B. longum subsp. infantis groups and control group was extracted by centrifugation, rinsed three times in
saline (0.9% NaCl), and then resuspended in saline before gavage. To counteract the sucrose impact, the
control group received the same care as the B. longum subsp. infantis group. The same strain intervention
group included all the pups from the same maternal mice. If there were less than eight puppies in each litter
for females and males, the pups were combined. Even though they were grouped, maternal mice continued
to feed their pups from separate litters. All the 3-week-old mice were sacrificed. Feces were collected before
being sacrificed and stored at -80 °C with 30% glycerin. A blood sample (100 μL), mesenteric lymph nodes
(MLNs), and colon were collected. Part of the colon was fixed in 4% formalin for immunofluorescence (IF).
MLNs were used for flow cytometry (FCM). The materials and reagents used are listed in the
Supplementary Materials.

MLNs were pulverized and passed through a 200-mesh cell sieve before adding an FC block agent (Miltenyi
Biotec, Bergisch Gladbach, Germany). Cells were collected and antibodies of CD3, CD4, and CD19 (Abcam,
Cambridge, UK) were added according to the instructions. The tube was incubated at 4 °C in the dark for
30 min, then supplemented with flow staining buffer, and centrifuged at 4 °C, 400 g for 5 min. The sediment
was mixed with flow staining buffer before being analyzed using flow cytometry.

The colon tissues were fixed in 4% paraformaldehyde and subjected to IF test. The slices were dehydrated
through gradient ethanol and then washed twice with PBS. Primary and secondary antibody staining were
performed on the slices after the antigen was repaired. The slices were sealed with anti-fluorescence
quenching agents after being washed with PBS three times. IF was automatically scanned with a 3D