And rest them overnight within a 37 five CO2 IGFBP-6 Proteins site incubator. 5.2 Transfer cells to a 15 mL tube and centrifuge for ten min at 500 g at RT. 5.three Aspirate supernatant, resuspend cells and add one mL of culture medium. five.4 Count the cells and change concentration to a hundred 106 cells/mL. five.5 Add one hundred L control mix for the appropriate wells of the non-tissue culture treated 96-well round bottom plate (3788, Corning). 5.6 Add a hundred L stimulation mix on the right wells on the 96-well plate. 5.7 Then include a hundred L cell suspension. five.8 Incubate for 4 h in a 37 5 CO2 incubator. 5.9 Put plate on ice for 15 min just after incubation. 5.ten Centrifuge plate for 5 min at 700 g at four . 5.11 Aspirate supernatant, resuspend cells in 200 L flow cytometry buffer and centrifuge plate again for five min at 700 g at 4 . five.12 Aspirate supernatant, resuspend cells in 50 L flow cytometry buffer containing a pretitrated appropriate level of surface staining mix. 5.13 Incubate for 30 min at four , shaking, protected from light. 5.14 Add 150 L flow cytometry buffer and centrifuge at 700 g at four for 3 min. five.15 Aspirate supernatant and include 100 uL of Cytofix/Cytoperm reagent (554722, BD Biosciences) to just about every nicely and resuspend by pipetting three times up and down. five.16 Incubate for 20 min at RT protected from light. 5.17 Add one hundred L movement cytometry buffer and centrifuge at 700 g at 4 for three min. five.18 Aspirate supernatant and include 50 L intracellular staining mix prepared in 1perm/wash and resuspend by pipetting 3 instances up and down. five.19 Incubate for thirty min at 4 , shaking, protected from light. 5.twenty Include 150 L 1perm/wash to every properly and centrifuge for 5 min at 700 g at four .Writer Manuscript Author Manuscript Writer Manuscript Writer ManuscriptEur J Immunol. Writer manuscript; offered in PMC 2022 June 03.Cossarizza et al.Page5.21 Aspirate supernatant, add 200 L 1perm/wash to each nicely and centrifuge for five min at 700 g at 4 . 5.22 Aspirate supernatant and resuspend cells in one hundred L movement cytometry buffer and analyze by movement cytometry cell sorting inside the sought after format. Note: protocol adapted from Lamoreaux et al. 421.Author Manuscript Author Manuscript Writer Manuscript Author Manuscript6 Monoclonal antibodies 6.one Surface staining:BD Biosciences: CD4 BUV 395 (SK3), CD45RA BV421 (HI100), CCR7 BUV395 (150503), CD45RA BV650 (HI100), CXCR5 Alexa Fluor488 (clone RF8B2), CD25 APC (clone 2A3) CD161 FITC (DX12). eBioscience: CD3 PE (UCHT1), KLRG1 AF488 (clone 13F12F2), CD4 PerCP-eFluor 710 (clone SK3), CD127 PECy7 (clone eBioRDR5), CD27 APC-eFluor 780 (clone O323), CD107a FITC (clone H4A3) Biolegend: CD27 APC-Fire 750 (O323), CCR6 Alexa Fluor647 (clone G034E3), CCR7 BV421 (clone G043H7), CX3CR1 FITC (clone 2A9), CCR4 BV421 (L291H4), CD28 Alexa Fluor 700 (CD28.2), CD127 BV650 (A019D5).R D Methods: CXCR3 PE (clone 49801)Sanquin: CD28 FITC (15E8)six.two Live/dead exclusion dyes: Live/dead fixable dyes (Thermofisher) or Fixable MNITMT Autophagy viability dye (eBioscience); we here use Fixable viability dye eFluor 506 (eBioscience). six.3 Intracellular stainings:BD Biosciences: IL-4 PE (3010.211), IFN BUV395 (B27), granzyme B Alexa Fluor700 (clone GB11), IL-2 PE (clone 5344.111), IL-10 BV650 (JES3D7), TNF- Alexa Fluor700 (clone MAb11), Perforin BV421 (clone B-D48), Hobit (clone 5A); eBioscience: IL-21 eFluor 660 (eBio3A3-N2), Eomes PerCPeFluor 710 (WD1928), Helios PE-Cy7 (22F6), IFN- APCeFluor 780 (clone 4S.B3), FoxP3 PE (clone PCH101), T-bet PE-Cy7 (clone 4B10) Biolegend: IL-17A BV421 (BL168), IL22 PE (BG/IL22), Anti-IgM PE (clone ma-69)7 Movement cytomete.
