Journal: Current Protocols
Article Title: Analyzing Pathophysiology and Immune Cells and Their Cytokines and Mediators in Precision‐Cut Slices of the Murine Lung
doi: 10.1002/cpz1.70087
Figure Lengend Snippet: Possible gateways for PCLS analysis. PCLSs can be analyzed in many ways. Among those, four are depicted here: ( A ) Paraffin embedding of PCLSs and (immuno‐)histochemical staining of lung sections. PCLSs are removed from conditioning medium and put into a biopsy cassette, fixed in 10% formalin‐PBS solution, dehydrated, and embedded in paraffin. The paraffin‐embedded PCLSs are cut into 4‐µm‐thick sections that can be used for different purposes (like immunohistochemistry). Exemplary H&E staining for visualization of a lung tumor, inflammation, and histology is depicted. ( B ) Transcriptome analysis of mRNA derived from cultured PCLSs. After conditioning, the PCLSs are put in RNA stabilization reagent for storage until further processing. Homogenization of the PCLSs is achieved by a tissue homogenizer using QIAzol and two metallic beads. RNA is extracted from the homogenized solution and further processed into cDNA to be analyzed by quantitative real‐time PCR (qPCR). ( C ) Conditioned PCLS–derived single‐cell analysis by flow cytometry. After conditioning, at least six PCLSs (dependent on PCLS size) are pooled, digested enzymatically with collagenase/DNase solution, and pushed through a 40‐µm cell strainer to generate a single‐cell suspension. The single‐cell suspension is subsequently stained with specific FACS antibodies to meet the individual needs for analysis and can be analyzed using a flow cytometer. An exemplary T‐cell analysis of a PCLS‐derived single‐cell suspension is depicted. ( D ) Cytotoxicity assay performed with PCLS cell culture medium: 7 × 10 3 LL/2‐luc‐M38 (LL/2) cells were cultured in a 96‐well plate at 37°C, 5% CO 2 , in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% l ‐glutamine. After 24 hr, the medium is removed, and the wells are washed with PBS and incubated with cell culture medium, 50% PCLS culture medium, or 50% CTLL2 culture medium at 37°C, 5% CO 2 . After 24 hr, the medium is again removed, and the wells are washed with PBS. Subsequently, 150 µg/ml luciferin is added, and after 25 min at 37°C, 5% CO 2 , fluorescence intensity can be evaluated. The fluorescence intensity for the evaluation of the depicted results was measured using a Centro XS³ LB 960 Microplate Luminometer (Berthold Technologies). For calculation of the results, a log‐log regression analysis was used. The results are depicted as degree of cytotoxicity, corresponding to the percentage of reduction of cells treated with PCLS and CTLL2 conditioned medium compared to LL/2 cells incubated in DMEM. For each group, mean with standard deviation is depicted, and each dot represents an individual sample.
Article Snippet: Murine LL/2‐luc‐M38 cell line (LL/2 cells; Bioware cell line, Caliper Life Sciences) LL/2 cell medium (see recipe), 37°C PBS, without calcium or magnesium (Gibco‐Invitrogen, cat. no. 14190) 30% to 50% (v/v) PCLS conditioned medium (PCLS‐CM), 37°C 30% to 50% (v/v) CTLL2 conditioned medium (CTLL2‐CM), 37°C Luciferin (Promega) 96‐well white‐walled plates, sterile (Berthold Technologies, cat. no. 24910) Adhesive foil (Berthold Technologies, cat. no. 55590) Centro XS³ LB 960 Microplate Luminometer (Berthold Technologies) NOTE : To prepare the PCLS‐CM, first culture PCLSs (see Basic Protocol) for 48 hr in PCLS medium (see recipe).
Techniques: Staining, Immunohistochemistry, Derivative Assay, Cell Culture, Homogenization, Real-time Polymerase Chain Reaction, Single-cell Analysis, Flow Cytometry, Suspension, Cell Analysis, Cytotoxicity Assay, Incubation, Fluorescence, Standard Deviation
