Immunophenotyping test v2 1/Apr/2023 RIKENMPP_030_002 test 3 1 testSt test modification

This test differentiates immune cell sub-population in mouse splenocyte via flow cytometry.

These procedures are applicable to the mouse phenotyping platform at the RIKEN BRC. The tests should be performed by trained staff members who understand the safety and hygiene rules required by RIKEN. Any queries, comments, or suggestions, relating either to this SOP in general or to a specific problem encountered during a procedure, should be addressed to the Blood Tests Unit Leader.

Testing is carried out in accordance with common animal handling procedures. Coveralls (in the rearing facility) or laboratory coats (in the laboratory facility) must be worn with gloves at all times in the work area. If animals are dropped on the floor, treat them in accordance with the safety and hygiene guidelines of the rearing facility at RIKEN. If something unexpected happens during the primary screening, it must be quickly reported to the Blood Tests Unit Leader and recorded.

Measure the cytometer setup and tracking (CS&T) Beads, before experiment according to the manufacturer's manual to ensure quality control.

Scissors and forceps for biopsy Precision balance Plate shaker Refrigerated centrifuge Automated cell counter (Countess; Thermo Fisher Scientific) Flow Cytometer (BD LSRFortessa; BD Biosciences) FlowJo software (TreeStar)

96-well U-bottomed plates (Falcon) 5 ml tubes 12x75 mm, 5ml tubes (Falcon) Cell strainers e.g. Nylon mesh Slides for cell counter (Chamber slides; Thermo Fisher Scientific) FBS (Fetal Bovine Serum) HBSS with Mg2+, with Ca2+ PBS without Mg2+, without Ca2+ Digestion enzyme (Collagenase D; SIGMA) 10x stock solution: 2000 Mandl Unitis/ml collagenase D in enzyme buffer (see in below), aliquoted and stored at -20℃ DNAse I (SIGMA) 10x stock solution: 0.1 mg/ml DNAse I in enzyme buffer (see in below), aliquoted and stored at -20℃. Cell lysis buffer (10x RBC lysis buffer; eBioscience) HEPES (pH 7.2) Fc blocking antibody (Mouse TruStain fcX antibody; Biolegend) Antibodies (see Appendix (1)) Antibody for live/dead discrimination (SytoxBlue; Invitrogen) Brilliant Stain buffer (BD Horizon)

Reagent preparation Enzyme buffer (Stable for up to 1 month in the fridge) ・HBSS with Ca2+ and Mg2+ ・2% FBS (v/v) ・10mM HEPES 1x Enzyme cocktail (require 3 ml per each spleen) ・Enzyme buffer ・Collagenase D (200 Mandl Units/ml) ・DNAse I (0.01 mg/ml) FACS buffer (Stable for up to 1 month in the fridge) ・PBS without Ca2+ and Mg2+ ・EDTA 2mM ・10mM HEPES ・2% FBS (v/v) 1x RBC lysis buffer ・Prepare a 1x solution in ddH2O from lysis buffer. Antibody cocktails for panel A and B (require 50 ml per each sample) ・Protect antibody and prepared cocktails from direct light. ・Panel A antibodies are dilute with FACS buffer and Panel B antibodies are dilute with Brilliant Stain buffer (see Appendix (1)). Spleen collection Collect the spleen from euthanized mice. Measure the collected spleen weight by precision balance. Place the spleen into a 5 ml tube with 3 ml of 1x enzyme cocktail on ice. Spleen dissociation and digests Mince into pieces using scissors, place on ice until all samples are minced. Incubate in a 37℃ water bath for 20 min. Cell suspension is filtered through a nylon mesh into 12x75 mm tube. Spin at 500 x g in a swing bucket rotor for 5 min at 10℃. Decant the supernatant, rack the tubes to resuspend the pellet. Add 2 ml FACS buffer, mix well by vortexing and keep on ice. Cell counting Take 10 ml for the counting step from cell suspension. Dilute 10 ml of cell suspension 100 times with FACS buffer. Dye the diluted cell with same amount of trypan blue. Count the live cell by automated cell counter. Calculate the concentration in cells per ml. Pipette the volume containing approximately 4 million cells per well to a 96 well plate in horizontal fashion starting from A1 onwards for panel A staining. Add 100 ml of FACS buffer per cell, centrifuge for 2 min, 510 x g at 4℃, discard supernatant and keep in ice. Red blood cell lysis, blocking and staining Remove plate from ice and add 50 ml of 1x RBC lysis buffer (at room temperature) to each cell pellet. Vortex the edges of the plates and incubate for 1 min at room temperature. Return to ice and add 100 ml of FACS buffer (to stop lysis) to each well. Centrifuge for 2 min, 510 x g at 4℃, discard supernatant and resuspend in 100 ml of FACS buffer. Again centrifuge for 2 min, 510 x g at 4℃ and discard supernatant. Resuspend in 100 ml of Fc blocking antibody (diluted 1/50) and incubate on ice for 10 min. Centrifuge the plate, discard supernatant and resuspend in 50 ml of antibody cocktail in appropriate wells for individual panels followed by incubation on ice and in the dark for 60 min. After incubation, add 100 ml of FACS buffer and centrifuge, discard supernatant. Resuspend the pellet in 200 ml of read buffer (SytoxBlue diluted 1:10000 in FACS buffer). Measurement and analysis Follow the cytometer (LSRFortessa) manual when measure the sample. Set up the cytometer to aim acquire 300,000 viable events (live cell) for each of panel A and B, save the data of all cells. Using FlowJo software, analyze the data following gating strategy (see Appendix (2) and Appendix (3)).

Antibody cocktails panel and dilution (panel A and B) [Appendix (1)] Parameter and gating steps (panel A) [Appendix (2)] Parameter and gating steps (panel B) [Appendix (3)]

[2023.4.1] For v2.1.1, "Brilliant Stain buffer" was added to "supplies". Updated the "Appendix (1)". Appendix (1): CD23 fluoresence was changed from CF594 to BV605. [2021.11.16] For v2.1, Updated the "Appendix (2)". Appendix (2): "CD4T" was deleted. Units for "Treg" and "CD4Th" were changed from "% of CD4T" to "% of T". [2021.11.16] For v2.0.1, Updated the "Appendix (1)" and "(3)". Appendix (1): "CD23" antibody was added to panel B. Appendix (3): Parameter for B cell subsets were added. [2020.12.1] A new version of the Immunophenotyping test (Ver. 2) was added.