Lymph nodes were collected from Rag1−/− B3K506 and Rag1−/− B3K508 (20 (link)) TCR transgenic mice and hand-mashed into a single cell suspension. Aliquots were stained with allophycocyanin-labeled CD4 (RM4–5; Tonbo biosciences) antibody and analyzed with Fluorescent AccuCheck counting beads (Invitrogen) to assess CD4+ T cell numbers and purity using a LSR II (BD biosciences) flow cytometer. For imaging experiments, 106 TCR transgenic cells were transferred into B6 mice 24 h before infection. 105 TCR transgenic cells were transferred into B6 mice for flow cytometry experiments examining the initial three d following Lm infection, while 3×103 TCR transgenic cells were transferred for experiments examining the response at seven d post-infection with Lm or influenza.
Fluorescent accucheck counting beads
Manufactured by Thermo Fisher Scientific
Fluorescent AccuCheck counting beads are a type of laboratory equipment used for accurate cell counting. The beads are fluorescently labeled and can be detected and quantified using flow cytometry or fluorescence microscopy. The core function of these beads is to provide a reliable and consistent method for enumerating cells or other particles in a sample.
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2 protocols using fluorescent accucheck counting beads
1
Adoptive Transfer of TCR Transgenic T Cells
2
Flow Cytometry Lymphocyte Quantification
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All antibodies were from eBioscience (San Diego, CA, USA), Biolegend (San Diego, CA, USA), or Tonbo (San Diego, CA, USA). All tetramers were obtained from the NIH tetramer facility (Atlanta, GA, USA). The tetramers were made by conjugating the biotin-labeled monomers with PE-labeled streptavidin (Prozyme, Hayward, CA, USA) according to protocols from the NIH tetramer facility. All flow cytometry data were acquired by BD LSR Fortessa analyzers (BD Biosciences) and were analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Fluorescent AccuCheck counting beads (Invitrogen) were used to calculate the total numbers of live lymphocytes.
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