DURAClone IF Monocyte Activation Protocol

This protocol is for reference purposes only.  It may be necessary to adapt the method for specific research needs.

Materials

KIT BOX CONTENTS

25 tests of the DURAClone IF Monocyte Activation Antibody Panel 

NOTE: Do not store the reagent tubes in the refrigerator; do not freeze/thaw the tubes. Minimize the exposure of the tubes to light, especially during processing and incubation of sample(s) prior to acquisition.

MATERIAL REQUIRED BUT NOT SUPPLIED

Blood collection tube containing sodium heparin anticoagulant

Calibrated pipettes

Vortex mixer

PerFix-nc Cellular Staining Preparation Kit (Part Number B10825):

  • Buffer 1, fixative reagent
  • Buffer 2, permeabilizing reagent
  • Buffer 3, final solution, 10X concentrate.  Dilute to 1X prior to use. 

Flow cytometer equipped with the following lasers and detectors:

  • FSC/SSC
  • 405 nm: 430 – 470 nm, 530 – 570 nm
  • 488 nm: 560 – 600 nm
  • 633 nnm: 715 – 735 nm

Sheath fluid

Flow cytometer calibration beads

Staining Procedure

SAMPLE PREPARATION FOR WHOLE BLOOD  

  1. 50 μL of activated blood to an appropriately labeled test tube.

  2. Add 5 μL of PerFix-nc Buffer 1. Vortex until red pellet is dissociated. Incubate 15 minutes at room temperature.

  3. Add 300 μL of PerFix-nc Buffer 2. Vortex.

  4. Transfer the contents into one tube of the DURAClone IF Monocyte Activation Antibody Panel.

  5. Vortex the tubes at high speed for 6-8 seconds. Incubate 15 minutes at room temperature. Protect from light.

  6. Add 3 mL of PerFix-nc Buffer 3. Vortex.

  7. Sample is ready for acquisition. Set the discriminator on the FS parameter to a value low enough to assure leukocytes are not excluded from acquisition.

Analysis

  1. Create a CD45-Krome Orange vs. SSC dot plot. Create a region to encompass the CD45+ leukocytes.

  2. Create a CD14-Pacific Blue vs. SSC dot plot. Apply the CD45+ gate to this plot. Create a region to encompass the CD14+ monocytes.

  3. On the CD45+ CD14+ monocytes assess the expression of HLA-DR-PE and TNFα-Alexa Fluor-700 by creating a TNFα-Alexa Fluor-700 vs HLA-DR-PE dot plot and a quadrant gate to differentiate subsets: HLADR+ TNFα-, HLA-DR+ TNFα+, HLA-DR- TNFα+ and HLA-DR- TNFα- cells.