In vitro TFH stimulation assay

In Vitro TFH stimulation Assay

Total splenocytes were isolated from immunized mice as described above, and total lymphocytes were resuspended in complete IMDM medium (Gibco). To measure cytokine production, total splenocytes were treated total lymphocytes with PMA (50ng/mL) and Ionomycin (1μg/mL) for 4 hours at 37ºC and 5% CO2. GolgiPlug (BD Biosciences) was added to the culture medium (concentration = 1:1000) after 1 hour of culture. At the end of the stimulation period, cells were fixed and stained for flow cytometry using previously described methods [Chen et al., 2021]. Notably, we used a specific clone of rat anti-mouse CXCR5-biotin (Biolegend, clone #L138D7) that can stain intracellular CXCR5 after paraformaldehyde fixation.

Flow Panel:

Surface Stain

Live/Dead-efluor780 (Thermofisher# 65-0865-18), Dilution 1:1000

PD1-BV785 (Biolegend #135225), Dilution 1:100

Postfixation Step #1

CD4-BUV737 (BD Biosciences #612844), Dilution 1:200

CXCR5-Biotin (Biolegend #145503), Dilution 1:300

IFNg-BV605 (Biolegend #505839), Dilution 1:200

IL4-FITC (Invitrogen, #11-2042-82), Dilution 1:100

IL13-PerCPe710 (Invitrogen # 46-7133-82), Dilution 1:100

IL21-PE (Invitrogen 12-7211-82), Dilution 1:100

Foxp3-PeCy7 (Tonbo [now cytek], #60-5773-U025), Dilution 1:200

CD40Lg-APC (Biolegend # 106510), Dilution 1:100

CD44-AF700 (Invitrogen # 56-0441-82), Dilution 1:200

Postfixation Step 2

SA-BV421 (Biolegend # 405225) , Dilution 1:400

Data were acquired on an Aurora Spectral Flow Cytometer