Engineer s micrometer

Mice were painted with 25 µl 0.15% 2,4-dinitrofluorobenzene (DNFB; Sigma-Aldrich) in 1:4 olive oil:acetone (OOA) mixture on the dorsal side of both ears for three consecutive days (days 0-2). On day 5, mice were euthanized and ear thickness was measured on both ears using an engineer's micrometer (Mitutoyo, Tokyo, Japan). Ear draining (d)LN were excised and analyzed by flow cytometry as described above.

Delayed-type hypersensitivity responses are inhibited in association with ACAID induction [22 (link)–26 (link)]. DTH assays were performed in a similar manner as previously described [17 (link)]. Briefly, left ear pinnae were injected intradermally with ∼500 μg of CII in 20 μl. Right ear pinnae were injected with 20 μl of 10 mM acetic acid (internal control). Seven days after the subcutaneous immunization with CII/CFA, DTH assays were performed. The engineer's micrometer (Mitutoyo, Japan) was used to perform ear swelling measurements before and 24 h after CII injection. The results were measured as: specific ear swelling = (24 h measurement − 0 hr measurement) for left ear − (24 h measurement − 0 hr measurement) for right ear. Results after 48 h were calculated in a similar manner: specific ear swelling = (48 h measurement − 0 hr measurement) for left ear − (48 h measurement − 0 hr measurement) for right ear.

Ts cell-derived EVs or B1 cell-produced EVs were administered to the Cas-immunized animals intradermally, intraperitoneally, intravenously or per os in equal doses (see above) at the peak of the allergic response, i.e., 24 h post-challenge. Ear thickness was measured up to 120 h after challenge by a blinded observer, unaware of the experimental protocol, using an engineer’s micrometer (Mitutoyo, Kawasaki, Japan). Otherwise, mice, 5 days prior to active immunization, had been injected intravenously with 10% suspension of syngeneic erythrocytes conjugated with Cas antigen, and subsequent ear swelling was elicited as above 5 days after immunization.

Mice were exposed to a single dose of 80 mJ/cm² of UVB. A Waldmann UV236B irradiation system equipped with two fluorescent CF-L 36 W/UV6 light tubes (emission range, 280–360 nm; peak, 324 nm; Waldmann Medizintechnik, Villingen-Schwenningen, Germany) was used for UVB irradiation, at a mean irradiance of 2.20 mW/cm² at a distance of 15 cm and the irradiance of exposure was monitored by a calibrated Waldmann photometer. The applied UVB dose corresponded to approximately one minimal erythema dose, as determined by exposure to a series of UVB doses at the increments by factor of 1.4, as previously described^{50 (link)}. To study the chronic effects, mice were exposed to repetitive UVB doses on alternate days for 23 days at a dose of 80 mJ/cm² for 11 days and then at a dose of 220 mJ/cm² from day 12 to 23 by using the same irradiation system. Skin inflammation was monitored by measuring the DSFT of dorsal skin with an engineer’s micrometer (Mitutoyo Corporation) and mice were killed 6 or 24 h after last UVB exposure for tissue collection. Noninvasive skin pigmentation was determined by skin reflectance spectroscopy on shaved dorsal skin with a DermaSpectometer (Cortex Technologies, DK).

On the third day of drug treatment, mice were actively sensitized to trinitrophenol (TNP) by topical application of 150 μL of a 5% picryl chloride (PCL, recrystallized 2,4,6-trinitrophenyl chloride, Chemica Alta, Edmonton, Alberta, Canada) dissolved in a 1:3 mixture of acetone and ethanol on the shaved abdominal skin. Five days later, mice were challenged to elicit CHS ear swelling by topical application of 10 μL of a 0.4% PCL solution in a 1:1 mixture of acetone and olive oil on both sides of both ears. At 2 (in the case of active CHS) and 24 h later, the induced ear swelling was measured with an engineer’s micrometer (Mitutoyo, Tokyo, Japan). The averaged results of the ear-thickness increase in each of the five mice in each group, after subtracting the ear-thickness increase in non-sensitized but challenged littermate mice, were expressed as delta +/− standard error of the mean (SEM). Otherwise, naive mice were similarly sensitized with 5% PCL solution on the shaved abdomen, and 5 days later, draining lymph nodes and spleens were collected for isolating CHS effector cells that were then adoptively transferred (via intravenous route) to drug-treated mice on the 8th day of treatment. Immediately after CHS effector cell transfer, recipients were similarly ear challenged with 0.4% PCL solution, and the elicited CHS ear swelling was measured as above.

DTH and LAT assays were performed as described elsewhere^{43 (link)–46 (link)}. For DTH experiments, mice infected with LM-OVA or LM-PLP were immunized with PLP178-191/CFA 7 days post-infection. Ten days post-immunization, mice were challenged intradermally in the ear pinna with antigen or vehicle control. Ear thickness was measured 48 h later and swelling responses were calculated. For LAT experiments, mice infected with LM-OVA or LM-PLP were immunized with PLP178-191/CFA seven days post-infection, and at 10 days post-immunization, they were euthanized and spleens were harvested. CD4 T cells from both groups were isolated following magnetic bead sorting (Miltenyi Biotec) protocols. CD4 T cells combined with naïve irradiated splenocytes and PLP antigen were intradermally injected into the ear pinna of naïve mice. Ear swelling responses were measured 24 h and 48 h post injection. In both assays, ear thickness was measured using an engineer’s micrometer (Mitutoyo).