Immunophenotyping [LA_IMM_002]

Purpose

This test differentiates immune cell sub-populations via flow cytometry.

Experimental Design

• Age at test: Week 57
• Sex: Both

Equipment

• Scissors and forceps for biopsy
• Precision balance
• Calibrated single and multichannel pipettes
• Plate shaker
• Refrigerated centrifuge
• Flow Cytometer (capable of distinguishing a minimum of 8 colours per well)
• Cell counter equipment

Supplies

• 96-well U-bottomed plates (Falcon #353077)
• Low retention pipette tips for antibody solutions
• 5ml tubes
• 12x75mm tube
• Cell strainers e.g. 70-80µM Nytex
• CS (calf serum)
• HBSS with Mg²⁺, with Ca²⁺ (for enzyme buffer used for DNAse and Collagenase D digestions)
• PBS without Mg²⁺, without Ca²⁺ (for FACS buffer to be used in all steps subsequent to enzymatic digest)
• EDTA (0.5M stock; final concentration 2mM)
• Digestion enzyme (Collagenase D from Roche #11088858001) stock solution in enzyme buffer (see below), aliquoted and stored at -20℃
• DNAse I stock solution (Sigma, #DN25) in enzyme buffer (see below), aliquoted and stored at -20℃
• RBC lysis buffer (eBioscience #00-4300-54, 10X from manufacturer)
• HEPES (pH 7.2-7.4)

Procedure

This protocol requires several steps in the collection, preparation and analysis of the samples. Each one is detailed separately below.

Reagent preparation

Note that two different PBS solutions are required for the protocol below, one with Ca²⁺ and Mg²⁺, another without Ca²⁺ and Mg²⁺.

• FACS buffer (for all steps subsequent to enzymatic digest; stable for up to 1 month in the fridge):
  • PBS 1X without Ca²⁺/Mg²⁺
  • EDTA 2mM
  • 2% (v/v) CS
  • 10mM HEPES, pH 7.2-7.4
• Brilliant Stain Buffer (BD 563794; for all steps when two or more brilliant violet antibodies are used to prevent non-specific dye-to-dye interaction)
• Enzyme buffer (for DNAse and Collagenase D digestions; Stable for up to 1 month in the fridge):
  • HBSS 1X with Ca²⁺/Mg²⁺
  • 2% (v/v) CS
  • 10mM HEPES, pH 7.2-7.4
• Enzyme cocktail (working solution): 3 ml for each spleen, containing final concentrations of:
  • DNAse I: 100 µg/ml (from 1 mg/ml stock in enzyme buffer stored in single experiment aliquots at -20℃, do not freeze-thaw stock)
  • Collagenase D: 0.17-0.2 Wünsch unit/ml (from 2 Wünsch unit/ml stock in enzyme buffer stored in single experiment aliquots at -20℃, do not freeze-thaw stock)
  NOTE: To top up to the 3ml use enzyme buffer; any intermediate dilutions of the enzyme stock solutions should be prepared with enzyme buffer.
• RBC Lysis buffer: Prepare a 1X solution in ddH20 from 10X stock lysis buffer.
• Antibody cocktails for Panels A & B
  • Protect antibodies and prepared cocktails from direct light.
  • Final concentration of antibodies should be determined by titration to ensure saturating amounts of antibody are used. Appropriate amounts of antibodies can be mixed together from the manufacturer's stock solutions and stored for 1 week at 4℃ prior to dilution in FACS buffer immediately before use. Do NOT pre-mix BV antibodies. These should be added fresh to the diluted staining mixure.
  • Each sample will require 50 µl of diluted 1X antibody cocktail.
  • Dilute antibody cocktail to final working concentration with FACS buffer, or Brilliant stain buffer when two or more brilliant violet antibodies are used, to make the AB mix.
  • Antibody cocktails should be gently but thoroughly mixed to ensure homogeneity of the solutions.
• Antibody Panels
  Recommended antibody (marker) panels, Panel A for T, NKT and NK cells, Panel B for B, myeloid and NK cells are shown below, along with optional markers that used by RBRC.

• Read buffer / dead cell exclusion dye
  • SytoxBlue at 1:10000 concentration in FACS buffer

Other preparations on the day

• Bring RBC lysis buffer and stop solution to room temperature.
• Prepare wet ice box, label tubes, etc.

Spleen collection

• Collect the spleen from euthanized mice.
• Remove all fat from the spleen and weigh the organ on a petri dish (do not hydrate the organ before weighing it as this would lead to substantial errors in measurement).

Spleen dissociation / digests

Performing manual digests:

• Place weighed spleen in 5ml tube containing 1ml of Enzyme cocktail (working solution)
• Mince into fine pieces using small scissors, place on ice until all samples are minced.
• Add 2ml of Enzyme cocktail (working solution) to each tube and place in a 37℃ water bath for 20 minutes.
• Tricturate (pipetting vigorously up and down using a 1 mL pipetman) the mixture to break up clumps.
• Filter cell suspension:
  • through 70-80 μm Nylon mesh filter into a 12x75mm tube
• Spin at 500 x g in a swing bucket rotor for 5 min at 10℃. Decant the supernatant, rack the tubes or vortex to resuspend the pellet. Add 2ml FACS buffer, mix well by vortexing, take 10 µl for the counting step.

Note: 96 well plate centrifuge steps are: 1800rpm for 2 minute at 4℃

Cell counting

• Perform a cell count on an aliquot of the re-suspended cells (adjust concentration according to the cell counter method used).
NOTE:Dilutions for counting: 2 serial 1:10 dilutions (10µl cells + 90µl FACS buffer, then 10µl of the 1:10 dilution + 90µl buffer.)
• Note the cell count, correct for dilution and calculate the concentration in cells per µl.
• Cell count:
  • Performed before RBC lysis, pipette the volume containing approximately 4 million cells/well to a 96 well plate in horizontal fashion starting from A1 onwards for panel A staining.
• Do the same for panel B staining in separate wells leaving a few empty rows between the panels to avoid cross contamination.
• Top up to final volume of 100 µl using FACS buffer, centrifuge, discard supernatant and keep plate on wet ice.

Red blood cell lysis, blocking & staining

Red blood cell lysis:

• Remove plate from ice and add 50 µl of 1X RBC lysis buffer (at room temperature) to each cell pellet from the previous step.
• Pipette up and down 2-3 times to break up the pellet and ensure complete lysis. Alternatively, vortex the edges of the plates, then pipet quickly once to ensure resuspension is ideal for optimal lysis.
• Incubate for 1 minute at room temperature and then return to ice and add 100 µl of FACS buffer (to stop lysis) to each well.
• Centrifuge, discard supernatant and resuspend in 100 µl FACS buffer.

blocking & staining:

• Again centrifuge and discard supernatant and resuspend in 100 µl of 1:50 Fc block and incubate on ice for 10 min.
• Centrifuge the plate after incubation, discard supernatant and resuspend in 50 µl 1X AB mix in appropriate wells for individual panels followed by incubation on ice and in the dark for 30 to 60 min.
• Using Sytox Blue as live/dead discriminator:
  • Top up to 200 µl with FACS or Brilliant Stain buffer after incubation. Centrifuge, discard supernatant.
  • Resuspend the pellet in 200 µl of read buffer (Sytox Blue diluted 1:10000 in FACS buffer).

General Recommendations for Setting up Cytometer

Set up the analyser to aim acquire 300,000 viable events (live cells) for each of Panels A and B.

Notes

Parameters & Metadata