Apc mouse igg1 k isotype control

A total of 0.5 x 10⁶ PBMCs were stained with the following combination of antibodies: Anti-CD19-BV421 (clone HIB19), anti-CD20-PerCPCy™5.5 (clone L27), anti-CD21-PE (clone B-ly4), anti-CD27-APC (clone M-T271) and anti-CD45-V500 (clone HI30). Anti-CD19-BV421, anti-CD20-PerCPCy™5.5, anti-CD21-PE, anti-CD27-APC, anti-CD45-V500, PerCP-Cy5.5 mouse IgG1 k isotype control, BV421 mouse IgG1 k isotype control, V500 mouse IgG1 k isotype control, PE mouse IgG1 k isotype control, and APC mouse IgG1 k isotype control were purchased from BD Biosciences (Becton Dickinson, Franklin Lakes, NJ, USA). B-cell populations were identified based on the expression of the following markers: activated memory B-cells (CD19+ CD20+ CD21- CD27+ CD45+), resting memory B-cells (CD19+ CD20+ CD21+ CD27+ CD45+), tissue-like memory cells (CD19+, CD20+ CD21- CD27- CD45+), and resting naïve B-cells (CD19+ CD20+ CD21+ CD27- CD45+).
CD34+ hematopoietic progenitors in peripheral blood were enumerated using the Stem Cell Enumeration Kit (Becton Dickinson).
All flow cytometry analyses were performed using a BD LSRFortessa™ Flow Cytometer (Becton Dickinson) from the I2MC cytometry platform (CHU Rangueil, Toulouse, France) and BD FACSDiva™ Software (Becton Dickinson).

Directly-labeled monoclonal antibodies were used for flow cytometry APC-mouse IGg1K isotype control (#555751, BD Biosciences, Grenoble, France) and APC-mouse anti-human CD279 (#558694, BD Biosciences, Grenoble, France). Two hundred thousand cells of each cell line were labeled at room temperature in PBS/1% BSA, washed twice in PBS, and analyzed on a BD ACCURI C6 cytometer (Paris, France). Cancer lines were gated according to their forward-scatter and side-scatter properties and excluding debris and doublets.

The MSC-induced CECs from passage 2 or 3 were harvested, washed twice with PBS, incubated for 10 min with 2 mL TrypLE (Invitrogen) at 37 °C, and collected. Cells were then stained for MSC or CEC markers using the Miltenyi Human MSC Phenotyping Kit (130-125-285; Miltenyi Biotec Ltd., Auburn, CA, USA) and antibodies against CD29 and CD166. 5 µL of each antibody cocktail was added at room temperature for 30 min. The cells were then washed twice by adding 3 mL flow cytometry buffer and centrifuging at 300× g for 10 min. The cells were then run through a Becton Dickinson FACSCanto I flow cytometer and the flow cytometry plots were analyzed using FACSDiva software. Antibodies used to detect CD29 and CD166 were as follows: PE Mouse Anti-Human CD166 (559263), APC Mouse Anti-Human CD29 (559883), PE mouse IgG1, K Isotype control (555749), APC mouse IgG1, K Isotype control (555751; BD Biosciences, San Jose, CA, USA).