Introduction

Lipopolysaccharide (LPS) is a strong activator of immune cells including B cells, monocytes, macrophages, and other LPS reactive cells. These immune cells require an immunogenic stimulator such as LPS to produce cytokines. For example, monocytes activated by LPS will secrete interleukin-6 (IL-6), interleukin-10 (IL-10), or TNF-α. Below is a protocol for immune cell stimulation with LPS.


Materials

  1. T25 or T75 sterile flasks with vented caps (e.g., Nunc EasYFlask Cell Culture Flasks, T25, filter, Cat. No. 156367)
  2. RPMI 1640 medium (e.g., BenchStable RPMI 1640 Medium, Cat. No. A4192301)
  3. Fetal Bovine Serum (e.g., Gibco Fetal Bovine Serum, Cat. No. 26140087, or Fetal Bovine Serum One Shot format, Cat No. A3160401)
  4. L-glutamine (e.g., 200 mM L-Glutamine, Cat. No. 25030149)
  5. Optional: penicillin-streptomycin (e.g., Gibco Penicillin-Streptomycin, Cat. No. 15140148)
  6. Lipopolysaccharide (LPS) (e.g., eBioscience Lipopolysaccharide Solution, Cat. No. 00-4976-93)
  7. Cell scraper (e.g., Nunc Cell Scrapers, Cat. No. 179693)


Procedure

  1. Using aseptic techniques under sterile conditions, isolate PBMC from whole blood or lymphoid tissue (See supplemental protocol A or B below, respectively).
  1. Prepare complete RPMI 1640 medium by supplementing RPMI 1640 medium with fetal bovine serum to a final concentration of 10% and 2 mM L-glutamine (if using medium not currently supplemented with GlutaMAX). Bring medium to 37°C. Optional: Supplement media with 1% penicillin-streptomycin (5,000 units/mL).
  1. Resuspend cells to a concentration of 3 x 106 cells/mL in complete RPMI 1640 medium. Prepare the volume needed based on the flask size you will be using (T25 or T75).
  1. Prepare two culture flasks: one labeled ‘stimulated’ (activated) and the other labeled ‘non-stimulated’ (non-activated).
  1. Divide the cell solution (prepared in step 3) evenly into the prepared flasks.
  1. Add lipopolysaccharide (LPS) at a concentration of 1X (2 µL/mL) into the stimulated (activated) flask. Cover both flasks with vented caps.
  1. Incubate both flasks for 1–3 days in a 5% CO2 incubator at 37°C.
  1. Harvest cells with a cell scraper.

Protocol tip:

Monocytes can be enriched by negative selection from PBMCs using the Monocyte Enrichment Kit (e.g., MagniSort Human pan-Monocyte Enrichment Kit, Cat. No. 8804-6837-74)


Supplemental protocol A: isolation of PBMC from whole blood

Materials

  1. Phosphate buffered saline (e.g., Gibco PBS (10X), pH 7.4, Cat. No. 70011044)
  2. 15 mL or 50 mL conical tube (e.g., Nunc 15 mL conical sterile centrifuge tubes, Cat. No. 339650)
  3. Ficoll-Paque® density separation medium
  4. Flow cytometry staining buffer (e.g., eBioscience Flow Cytometry Staining Buffer, Cat. No. 00-4222-26)

Procedure

  1. Dilute blood sample at least 1:1 with PBS in a conical tube.
  1. Underlay the diluted sample with a volume of Ficoll-Paque® medium that is equal to the original sample volume.
  1. Centrifuge at 400 x g for 20 minutes at room temperature with the brake OFF.
  1. Harvest PBMC located at the interface of the PBS and Ficoll-Paque® medium layers into a fresh tube.
  1. Fill the tube with PBS to wash the cells.
  1. Centrifuge the cells at 300–400 x g for 4–5 minutes at 2–8°C. Discard supernatant.
  1. Resuspend the cell pellet in an appropriate volume of flow cytometry staining buffer or buffer of choice, and perform a cell count and viability analysis.
  1. Centrifuge cells as in step 6, and resuspend in appropriate volume of complete RPMI 1640 so that the final cell concentration is 3 x 106 cells/mL.

Note:

As cells will be cultured, perform all steps using aseptic technique, and use buffers that do not contain azide.

Protocol tip:

Significantly improve the accuracy of assessing cell health and concentration from freshly harvested peripheral blood mononuclear cells with automated counters. See detailed protocol on how to count PBMC using the Countess II FL Automated Cell Counter.


Supplemental protocol B: isolation of immune cells from lymphoid tissue

Materials

  1. 60 x 15 mm cell culture dish (e.g., Nunc Cell Culture Petri Dishes, Cat. No. 150340)
  2. Plastic 3-mL syringe or two frosted glass microscope slides
  3. Cell strainer (nylon mesh)
  4. 15 mL or 50 mL conical tube (e.g., Nunc 15 mL conical sterile centrifuge tubes, Cat. No. 339650)
  5. Flow cytometry staining buffer (e.g., eBioscience Flow Cytometry Staining Buffer, Cat. No. 00-4222-26)

Procedure

  1. Harvest tissue (spleen; thymus; lymph nodes) into a cell culture dish containing 10 mL of Flow Cytometry Staining Buffer or buffer of choice. Tease apart into a single-cell suspension by pressing with the plunger of a 3 mL syringe. Alternatively, mash tissue between the frosted ends of two microscope slides using 10 mL of Flow Cytometry Staining Buffer.
  1. Place a cell strainer on top of a 15-mL conical tube. Pass cells from the cell culture dish through the cell strainer to eliminate clumps and debris.
  1. Centrifuge cell suspension at 300–400 x g for 4–5 minutes at 2–8°C. Discard the supernatant.
  1. Resuspend the cell pellet in an appropriate volume of Flow Cytometry Staining Buffer or buffer of choice and perform a cell count and viability analysis.
  1. Centrifuge cells as in step 3, and resuspend in appropriate volume of complete RPMI 1640 medium to the final cell concentration is 3 x 106 cells/mL.

Note:

Mechanical disruption of lymphoid tissue is generally sufficient to release cells into a single-cell suspension.

Note:

As cells are to be cultured, perform all steps using aseptic technique, and use buffers that do not contain azide.