A Mr. Frosty (Nalgene), CoolCell (Corning) or maybe a freezing apparatus at -80 to get a period of four to 24 h. one.13 Shop the vials until even further use in liquid nitrogen.Author Manuscript Author Manuscript Author Manuscript2 Thawing PBMC 2.one Thaw the vials by gently shaking within a 37 water bath, until tiny ice stays. two.two Transfer the contents of your vial to a 50 mL tube. two.three Add drop by drop, while gently shaking, 18 mL of cold thawing medium. 2.four Let the cell suspension rest for 20 min and centrifuge for ten min at 500 g. 2.five Aspirate supernatant, resuspend pellet in 50 mL washing medium and centrifuge for ten min at 250 g at four . two.six Aspirate supernatant, resuspend pellet in desired volume of movement cytometry buffer (for surface and intracellular stainings) or culture medium (for stimulations) and count cells.three Surface staining three.one Transfer as much as 2 106 PBMC to a 96-well round buttom plate (Greiner BioOne). three.2 Centrifuge the plate at 390 g at 4 for three min. three.three Aspirate supernatant and resuspend cells by gently vortexing the plate. 3.4 Add 30 L flow cytometry buffer containing a pretitrated appropriate volume of MC3R Molecular Weight tetramer for each properly (put together 1extra).Writer ManuscriptEur J Immunol. Author manuscript; accessible in PMC 2022 June 03.Cossarizza et al.Page3.5 Incubate for 30 min at four , shaking, protected from light. three.six Meanwhile prepare surface staining (including the live/dead exclusion dye) in a complete volume of 30 L flow cytometry-buffer for each properly (prepare 1extra). 3.7 Include 30 L surface staining combine, devoid of washing the cells, straight to the nicely and incubate for a further thirty min at 4 , shaking, protected from light. 3.8 Include 150 L flow cytometry buffer and centrifuge at 390 g at four for 3 min. three.9 Resuspend cells by gently vortexing the plate. three.ten Include 100 L flow cytometry buffer, and analyze by movement cytometry cell sorting while in the sought after format, or continue with the intracellular staining protocol. Note: Generally use appropriately titrated antibodies and tetramers, that’s typically not the concentration advised through the supplier. The ins and outs of titrating antibodies can be observed inside the publication of ACAT2 custom synthesis Lamoreaux et al. 421.Writer Manuscript Writer Manuscript4 Intracellular stainings of transcription variables and cytolytic molecules four.1 Following surface staining add 200 L Fixation/Permeabilization buffer. four.2 Gently resuspend the cells by pipetting up and down three occasions. four.three Incubate for 20 min at four , shaking, protected from light. 4.4 Centrifuge for five min at 700 g at 4 . four.five Aspirate supernatant and resuspend cells in 200 L flow cytometry buffer and centrifuge for five min at 700 g at four . four.6 Aspirate supernatant and resuspend cells by pipetting up and down three times in 50 L of the intracellular staining combine ready in Permeabilization Buffer. 4.seven Incubate 30 min at four , shaking, protected from light. 4.8 Include 150 L Permeabilization Buffer to each and every properly and centrifuge for five min at 700 g at four . four.9 Aspirate supernatant and resuspend cells in 200 L Permeabilization Buffer and centrifuge for five min at 700 g at four . four.ten Aspirate supernatant and resuspend cells in a hundred L flow cytometry buffer and analyze by flow cytometry cell sorting from the sought after format.Writer Manuscript Author Manuscript5 Cytokine staining five.1 Transfer PBMC into suspension culture flasks (690 190, Greiner) at 1 106 cells/mL in culture. medium (flask standing upright, or 45Eur J Immunol. Author manuscript; readily available in PMC 2022 June 03.Cossarizza et al.Pagetilted determined by volume).