Home › Immunology › Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment
Immunology JoVE (Open Access) Citable · DOI

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

DOI: 10.3791/59427-v
What you'll learn
  • ✓ Isolate and purify CD4+ T cells and alveolar macrophages from bronchoalveolar lavage fluid
  • ✓ Perform immunophenotyping of pulmonary immune cells using flow cytometry sorting
  • ✓ Quantify HIV DNA and RNA in lung reservoir compartments using ultrasensitive PCR
Protocol

Biopharma Insights We describe a method for processing bronchoalveolar lavage fluid and matched peripheral blood from chronically HIV-infected individuals on antiretroviral therapy to assess pulmonary HIV reservoirs. These methods result in the acquisition of highly pure CD4 T cells and alveolar macrophages that may subsequently be used for immunophenotyping and HIV DNA/RNA quantifications by ultrasensitive polymerase chain reaction.

Difficulty
advanced
Total time
~4–6 hours per patient sample (BAL collection + processing + sorting)
Biosafety
BSL-2

Steps

1
Isolate viable cells from bronchoalveolar lavage fluid

Process fresh BAL fluid to recover and enrich living cells for downstream analysis. Perform cell counting and viability assessment to ensure sample quality.

▶ 01:02
2
Sort BAL and PBMC populations by flow cytometry

Use fluorescence-activated cell sorting to isolate pure populations of alveolar macrophages and CD4+ T cells from whole BAL and matched peripheral blood mononuclear cells. Collect sorted populations for downstream analysis.

▶ 02:33
3
Verify alveolar macrophage immunophenotype by flow data

Review representative flow cytometry plots to confirm marker expression and purity of sorted alveolar macrophage and CD4+ T cell populations before HIV quantification.

▶ 05:48
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