Anti mouse igg3 alexa 488 antibody

Neutralizing antibody detection was performed as previously described [11 (link)]. Shortly, serum was heat-inactivated at 56 °C for 45 min and incubated with Cs (MOI of 0.5) at 37 °C for 30 min in a 1:10 final serum dilution. Next, confluent HeLa cells were infected with this serum-Cs mix by centrifugation at 900× g for 1 h at 37 °C. After an additional hour of incubation, cells were washed and incubated for 30 h. Then, cells were harvested and stained for flow cytometry evaluation of infection using the anti-chlamydia antibody clone ACI (LSBio, Seattle, WA, USA) and the secondary anti-mouse IgG3-Alexa 488 antibody (Southern Biotech, Birmingham, AL, USA). Cells were recorded on a Cytoflex flow cytometer using the CytExpert software (Beckman Coulter, Brea, CA, USA). Data analysis was performed with FlowJo version 10.5.3 (FLOWJO LLC, Ashland, OR, USA) as previously described [24 (link)]. Percent suppression was calculated for each animal using the following formula: $\% \mathrm{suppression}=100-\left(\frac{\% \mathrm{infection} \left[\mathrm{x} \mathrm{dpc}\right]}{\% \mathrm{infection} \left[-2 \mathrm{dpc}\right]}\right)\times 100$
where “x dpc” (days post challenge) is the day of the calculated percent of suppression.

Chlamydia trachomatis developmental cycle in pOECs from six individual animals was also monitored via flow cytometry as previously described [41 (link)]. In addition, the CLDN-4 protein expression was quantified in Ct- or MOCK-infected pOECs. These pOECs were washed with PBS, trypsinized and stained with Live/Dead Near infra-red. Next, cells were fixed and permeabilized with the eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (ThermoFisher). Then, cells were stained with the primary antibodies anti-Chlamydia antibody clone ACI (mouse IgG3, LSBio) and anti-CLDN-4 antibody (rabbit polyclonal, Abcam), and then with the secondary antibodies anti-mouse IgG3-Alexa 488 antibody (Southern Biotech) and anti-rabbit-Alexa 647 (Southern Biotech). Cells were recorded on a Cytoflex flow cytometer using the CytExpert software (Beckman Coulter, Brea, CA, USA). Data analysis was performed with FlowJo version 10.5.3 (FLOWJO LLC, Ashland, OR, USA). The gating hierarchy used for the differentiation of Ct-infected and MOCK-infected cells is shown in Supplementary Figure S2. The Ct gate was set based on MOCK-infected control cells.