And rest them overnight inside a 37 5 CO2 incubator. five.2 Transfer cells to a 15 mL tube and centrifuge for 10 min at 500 g at RT. five.3 Aspirate supernatant, resuspend cells and include 1 mL of culture medium. 5.four Count the cells and alter concentration to a hundred 106 cells/mL. five.five Include 100 L handle mix towards the right wells of a non-tissue culture handled 96-well round bottom plate (3788, Corning). five.6 Add one hundred L stimulation combine for the correct wells in the 96-well plate. five.7 Then include one hundred L cell suspension. 5.eight Incubate for four h in the 37 five CO2 incubator. 5.9 Place plate on ice for 15 min soon after incubation. five.10 Centrifuge plate for five min at 700 g at four . five.eleven Aspirate supernatant, resuspend cells in 200 L flow cytometry buffer and centrifuge plate yet again for 5 min at 700 g at four . 5.12 Aspirate supernatant, resuspend cells in 50 L movement cytometry buffer containing a pretitrated ideal quantity of surface staining combine. five.13 Incubate for 30 min at 4 , shaking, protected from light. five.14 Add 150 L movement cytometry buffer and centrifuge at 700 g at four for 3 min. 5.15 Aspirate supernatant and add a hundred uL of Cytofix/Cytoperm reagent (554722, BD Biosciences) to every single very well and resuspend by pipetting three BMP Receptor Proteins Formulation instances up and down. 5.sixteen Incubate for 20 min at RT protected from light. five.17 Add 100 L movement cytometry buffer and centrifuge at 700 g at 4 for three min. 5.18 Aspirate supernatant and add 50 L intracellular staining combine ready in 1perm/wash and resuspend by pipetting 3 occasions up and down. 5.19 Incubate for 30 min at four , shaking, protected from light. five.20 Add 150 L 1perm/wash to each effectively and centrifuge for five min at 700 g at four .Writer Manuscript Hydroxyflutamide Biological Activity Author Manuscript Author Manuscript Author ManuscriptEur J Immunol. Author manuscript; obtainable in PMC 2022 June 03.Cossarizza et al.Page5.21 Aspirate supernatant, include 200 L 1perm/wash to just about every well and centrifuge for five min at 700 g at four . five.22 Aspirate supernatant and resuspend cells in 100 L flow cytometry buffer and analyze by flow cytometry cell sorting within the wanted format. Note: protocol adapted from Lamoreaux et al. 421.Writer Manuscript Writer Manuscript Author 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 Systems: CXCR3 PE (clone 49801)Sanquin: CD28 FITC (15E8)6.2 Live/dead exclusion dyes: Live/dead fixable dyes (Thermofisher) or Fixable viability dye (eBioscience); we here use Fixable viability dye eFluor 506 (eBioscience). 6.three 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)seven Flow cytomete.
