Cell Surface CD74 Measurements.

Cell Surface CD74 Flow Cytometry Protocol

Required Materials

• Reagents:
  • Thawing/Stim Medium: RPMI 1640 medium supplemented with 5% FBS, 1% Glycine (2 mL of 100× stock), and 1% Pyruvate (2 mL of 100× stock)
  • Staining Solution: 1× PBS supplemented with 5% BSA
  • Antibodies/Dyes:
    • FITC-conjugated anti-CD74 (Clone: MB731; BD Biosciences)
    • APC-conjugated anti-CD11b (Clone: ICRF44; BioLegend)
    • 7-aminoactinomycin D (7-AAD; BD Biosciences)
• Equipment: Water bath (37°C), Centrifuge, Accuri C6 flow cytometer (BD), 96-well round-bottom plates or 5 mL flow tubes.

Step 1: Reagent Preparation

1. Prepare Thawing Medium: Combine RPMI 1640, 5% FBS, 2 mL of 100× glycine, and 2 mL of pyruvate. Warm to 37°C in a water bath prior to use.
2. Prepare Staining Solution: Dissolve BSA in 1× PBS to a final concentration of 5%. Keep chilled at 4°C.
3. Antibody Cocktail: Pre-dilute or prepare a cocktail of FITC-CD74 and APC-CD11b in Staining Solution according to manufacturer-recommended or pre-titrated volumes per test. Keep on ice and protected from light.

Step 2: PBMC Thawing & Washing

1. Thaw Cells: Retrieve frozen PBMC vials from liquid nitrogen. Immediately submerge the lower half of the vials in a 37°C water bath. Swirl gently until only a small ice crystal remains (approx. 1–2 minutes).
2. Dilute: Transfer the cell suspension slowly into a centrifuge tube containing 10 mL of pre-warmed Thawing Medium to dilute the DMSO.
3. Centrifuge: Spin the tubes at 300 × g for 5 minutes at room temperature.
4. Resuspend: Aspirate the supernatant without disturbing the pellet. Resuspend the cell pellet in 1–2 mL of room-temperature Thawing Medium.
5. Count: Take an aliquot to count live cells and determine cell viability.

Step 3: Cell Surface Staining

1. Aliquoting: Transfer approximately \(0.5 \times 10^6\) to \(1 \times 10^6\) viable PBMCs per sample into designated flow cytometry tubes or wells. Include extra tubes for single-stain controls (FITC only, APC only, 7-AAD only) and an unstained control.
2. Wash: Centrifuge at 300 × g for 5 minutes. Aspirate the supernatant.
3. Block/Resuspend: Resuspend each cell pellet in 50–100 µL of ice-cold Staining Solution (5% BSA acts as a blocking agent to reduce non-specific binding).
4. Antibody Incubation: Add the designated volumes of FITC-conjugated CD74 and APC-conjugated CD11b antibodies to the experimental samples. Add single antibodies to respective compensation control tubes.
5. Incubate: Mix gently by pipetting or tapping. Incubate for 20–30 minutes at 4°C in the dark.
6. Wash: Add 1–2 mL of ice-cold Staining Solution to each tube. Centrifuge at 300 × g for 5 minutes at 4°C and discard the supernatant.

Step 4: Viability Staining

1. Resuspend: Resuspend the stained cell pellet in 200–400 µL of Staining Solution.
2. Add 7-AAD: Add 5 µL of 7-AAD viability dye to each sample tube approximately 5–10 minutes before acquisition.
3. Incubate: Keep samples on ice and protected from light until analysis. Do not wash out the 7-AAD.

Step 5: Flow Cytometry Acquisition & Gating

1. Instrument Setup: Turn on the BD Accuri C6 flow cytometer and perform standard daily QC.
2. Compensation: Run unstained and single-stained control tubes to calculate and apply fluorescence compensation matrices (specifically matching FITC, APC, and 7-AAD spectral overlaps).
3. Data Collection: Acquire the MS and HC patient samples at a consistent flow rate, collecting a set number of target events (e.g., 50,000 total events per sample).
4. Gating Strategy:
   • Gate 1 (Cells): Plot FSC-H vs SSC-H to identify the main lymphocyte/monocyte PBMC population.
   • Gate 2 (Singlets): Plot FSC-H vs FSC-A to exclude cell doublets.
   • Gate 3 (Viable Cells): Plot 7-AAD vs FSC or SSC. Gate strictly on the 7-AAD negative population to isolate living cells.
   • Gate 4 (Target Population): Plot CD11b (APC) vs CD74 (FITC) on the viable singlet gate. CD11b is typically used to distinguish monocytes/myeloid subsets from lymphocytes. Quantify the Mean Fluorescence Intensity (MFI) or percentage of CD74+ expression across your targeted patient groups.