A Mr. Frosty (Nalgene), CoolCell (Corning) or possibly a freezing apparatus at -80 to

A Mr. Frosty (Nalgene), CoolCell (Corning) or possibly a freezing apparatus at -80 to get a period of four to 24 h. one.13 Store the vials until finally further use in liquid nitrogen.Writer Manuscript Writer Manuscript Author Manuscript2 Thawing PBMC two.one Thaw the vials by gently shaking within a 37 water bath, until very little ice remains. 2.2 Transfer the contents in the vial to a 50 mL tube. 2.three Insulin-like Growth Factor 2 (IGF-II) Proteins supplier Include drop by drop, though gently shaking, 18 mL of cold thawing medium. 2.4 Allow 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 10 min at 250 g at 4 . two.six Aspirate supernatant, resuspend pellet in wanted volume of flow cytometry Inhibitory checkpoint molecules Proteins MedChemExpress buffer (for surface and intracellular stainings) or culture medium (for stimulations) and count cells.3 Surface staining 3.1 Transfer up to two 106 PBMC to a 96-well round buttom plate (Greiner BioOne). three.two Centrifuge the plate at 390 g at 4 for three min. three.3 Aspirate supernatant and resuspend cells by gently vortexing the plate. 3.4 Include 30 L flow cytometry buffer containing a pretitrated acceptable quantity of tetramer for each well (prepare 1extra).Author ManuscriptEur J Immunol. Author manuscript; accessible in PMC 2022 June 03.Cossarizza et al.Page3.five Incubate for 30 min at 4 , shaking, protected from light. three.six Meanwhile prepare surface staining (like the live/dead exclusion dye) in the total volume of thirty L flow cytometry-buffer for every very well (prepare 1extra). 3.7 Add 30 L surface staining mix, devoid of washing the cells, directly in to the properly and incubate for any even further thirty min at 4 , shaking, protected from light. three.eight Include 150 L flow cytometry buffer and centrifuge at 390 g at four for three min. three.9 Resuspend cells by gently vortexing the plate. 3.ten Include a hundred L movement cytometry buffer, and analyze by flow cytometry cell sorting inside the preferred format, or carry on using the intracellular staining protocol. Note: Usually use appropriately titrated antibodies and tetramers, which is generally not the concentration advised by the supplier. The ins and outs of titrating antibodies can be identified inside the publication of Lamoreaux et al. 421.Author Manuscript Author Manuscript4 Intracellular stainings of transcription components and cytolytic molecules 4.one Soon after surface staining add 200 L Fixation/Permeabilization buffer. 4.two Gently resuspend the cells by pipetting up and down three occasions. four.three Incubate for 20 min at 4 , shaking, protected from light. 4.four Centrifuge for 5 min at 700 g at four . four.5 Aspirate supernatant and resuspend cells in 200 L flow cytometry buffer and centrifuge for 5 min at 700 g at 4 . 4.six Aspirate supernatant and resuspend cells by pipetting up and down 3 occasions in 50 L from the intracellular staining combine ready in Permeabilization Buffer. 4.7 Incubate thirty min at four , shaking, protected from light. four.eight Add 150 L Permeabilization Buffer to each and every very well and centrifuge for five min at 700 g at four . 4.9 Aspirate supernatant and resuspend cells in 200 L Permeabilization Buffer and centrifuge for 5 min at 700 g at four . four.ten Aspirate supernatant and resuspend cells in a hundred L movement cytometry buffer and analyze by flow cytometry cell sorting during the wanted format.Writer Manuscript Writer Manuscript5 Cytokine staining five.one 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; offered in PMC 2022 June 03.Cossarizza et al.Pagetilted depending on volume).