La concepción estructuralista de las teorías

2.3.1 Tissue digestion

Harvested tissues were kept in 5ml ice-cold RPMI until ready for processing. The tissues were then placed in 2ml of digestion buffer (RPMI 2% FCS with 0.8mg/ml Collagenase/Dispase (Roche), 0.2mg/ml Collagenase P (Roche) and 0.1mg/ml DNase1 (SIGMA)) and the organ structure was mechanically disrupted using scissors. The tubes were then incubated at 37°c with constant agitation for 20 minutes. The samples were repeatedly pipetted using p1000 during the incubation time to increase separation. After 20 minutes, the supernatant was removed from the samples and filtered through a 70µm cell strainer into 10ml ice cold MACS buffer (PBS, 1% BSA, 0.2mM EDTA), and then kept on ice. 2ml fresh digestion buffer was then added to each sample, which were incubated for 10 minutes at 37°c with constant agitation. Samples were pipetted regularly during the incubation to increase the separation of the cells. After 10 minutes, the samples were filtered through 70µm cell strainer into the 10ml ice cold MACS buffer and washed through with another 10ml MACS. Further digestion steps can be undertaken until all fragments are completely digested. The MACS buffer should now contain the entire cell fraction of the organs. The cells were then pelleted by centrifuging samples at 300xg for 4 minutes at 4°c. The resulting cells were then counted and stained in appropriate amount of antibodies as below.

Purified collagenase enzymes are used for this digestion in preference to traditional collagenase preparations taken from different fractions of bacterial supernatants because of the reduction in composition variability and the increase in stability of the preparation (Cavanagh et al., 1997). The collagenase fractions also present higher levels of endotoxins, which may cause activation of the immune cells, affecting the validity of the assay (Vargas et al., 1997, Eckhardt et al., 1999, Vargas et al., 1998). The selection of digestion method to study the stromal compartment is also based on the yield of cells required. The different collagenase enzymes can be classified based on their activity.

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Collagenase D has a much higher activity than Collagenase P. Because of this increased activity, Collagenase D is more successful at disrupting the bonds between the cells, allowing for better separation of the cell populations. However, use of this method also requires Collagenase Dispase enzyme to be used separately, adding time to the protocol. The strength of the enzymes might also compromise the preservation of some of the cell surface markers, thus making difficult in the assessment of the cells by flow cytometry and limiting the markers that can be used to differentiate the cells.

Collagenase P requires a slightly longer time to separate the cells, although this enzyme can be used in conjunction with the Collagenase Dispase enzyme, making this method comparable in duration to the Collagenase D digestion protocol. This milder digestion presents the advantage of better preservation of the markers, reducing the loss of cell surface markers necessary for the flow cytometric analysis. Collagenase P digestion was used throughout this thesis as the retention of cell surface markers was considered essential. Representative plots are shown in figure 2.1, indicating that collagenase P is far better at retaining cell surface gp38 expression that collagenase D.

Figure 2.1 Comparison of the lymph node stromal populations generated by different digestion methods. Comparisons of the classical lymph node stromal cell populations

achieved following different digestion protocols, with the results for Collagenase D digestion shown in (A) and the results from Collagenase P digestion shown in (B)

Gated on CD45-EpCAM- cells

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2.3.2 Embryological tissue digestion

Embryological lymph node anlage were isolated as outlined above and then digested for flow cytometry analysis. These organs were placed into 500µl digestion buffer (RPMI 2% FCS, Collagenase/Dispase 2.5mg/ml (Roche), DNase1 100µg/ml (SIGMA)) and incubated at 37°c with constant agitation for 30 minutes. These samples were pipetted several times during incubation to ensure thorough separation. 50µM EDTA was then added to each sample, which were incubated at 37°c for a further 5 minutes. Cells were centrifuged at 300xg for 4 minutes at 4°c. The resulting pellet was then resuspended in sterile RPMI (10% FCS, 1% GPS) and the samples were incubated overnight at 37°c in 5% CO2 on a 6 well plate. The following day the cells were scratched off the plate and washed

out of RPMI before staining in antibodies as outlined below.

2.3.3 Lymphocyte separation

Lymphocytes were prepared from the spleen by disruption and filtration through a 70μm cell strainer. The single cell suspensions produced were washed thoroughly in MACS buffer (PBS 1% BSA, 0.2mM EDTA). Erythrocytes were removed from the suspension by incubation for 5 minutes in 1ml red blood cell lysis buffer (SIGMA). Cells were then washed in PBS, before being centrifuged at 300xg for 4 minutes at 4°c. The resulting cell pellet was then cell pellet was then resuspended in MACS buffer for staining in antibodies as outlined below.

2.3.4 Preparation of in vitro cells for flow cytometry

Cells were removed from the incubators and were scratched off the plastic to prevent loss of cell surface markers. These cells were then washed out of media in sterile PBS at 300xg for 4 minutes at 4°c. The resulting cell pellet was then resuspended in MACS buffer for staining in antibodies as outlined below.

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2.3.5 Enrichment of stromal cell population using MACS™ beads

Lymphoid stromal cells are understood to make up between 1-5% of the total lymph node cellularity (Malhotra et al. 2012). In order to enrich this population and allow for better separation of the resulting stromal populations, the digested cells were incubated with CD45+ MACS (Miltenyi Biotec) beads at 10μl per 107 _{cells for 15 minutes at 4°c. LD columns (Miltenyi Biotec) were placed onto a}

Quadro MACS separator and then preactivated with buffer. Cells were then run through the columns the effluent collected. The columns were washed through twice to ensure all negative cells were washed through. The resulting cells were then centrifuged at 300xg for 4 minutes at 4°c. The resulting cell pellet was then cell pellet was then resuspended in MACS buffer for staining in antibodies as outlined below.

2.3.6 Staining

The cells were then stained for 30 minutes at 4°c in the dark in 100μl MACS buffer with antibodies diluted to the correct concentration as shown in table 2.1. Following washing in MACS buffer as above, the cells were then incubated in fixation/permeabilisation (1:4 fixation/permeabilisation concentrate:diluent, both from eBioscience) for 30 minutes at 4°c in the dark. These cells were then washed out of fixing solution using eBioscience wash buffer and resuspended in MACS buffer for acquisition. In order to ensure compensation was set correctly, compensation controls were used. For these controls, BD™ CompBeads were stained with single antibodies. Fluoresence minus one (FMO) controls were also used in order to eliminate false positives in the two-step staining protocols.

Cells were analysed using a CyAn™ ADP Analyzer (Beckman Coulter) with forward/side scatter gating to exclude non-viable cells. Data were then analysed using FlowJo v7 software (Tree Star). For cell sorting, stained ells were sorted using a MoFlo-XDP (Beckman Coulter). The purity of sorted stromal cell populations routinely exceeded 96%.

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Antibody (clone) Format Working Dilution Isotype Supplier

CD45 (30-F11) APC 1:200 Rat IgG2b eBiosciences

CD31 (390) FITC 1:200 Rat IgG2a eBiosciences

gp38 (ebio8.1.1) PECy7 1:200 Hamster IgG eBiosciences

ICAM-1/CD54 (YN1/1.7.4) PE 1:200 Rat IgG2b eBiosciences

VCAM-1/CD106 (MVCAM.A) PERCPCy5.5 1:50 RatIgG2a Biolegend

CD3ε (145-2C11) FITC 1:100 Hamster IgG BD Biosciences

B220 (RA3-6B2) eFluor 450 1:100 Rat IgG2a BD Biosciences

CD35 (8C12) biotinylated 1:100 Rat IgG2a BD Biosciences

CD4 (RM4-5) eFluor 450 1:50 Rat IgG2a eBiosciences

CD157 (BP-3) APC 1:200 Mouse IgG2b Biolegend

GL-7 (GL7) Alexafluor 647 1:100 Rat IgM eBiosciences

CD95/Fas (15A7) biotinylated 1:100 Mouse IgG1 eBiosciences

CD19 (eBio1D3) FITC 1:200 Rat IgG2a eBiosciences

Ter119 (ter-119) FITC 1:100 Rat IgG2a eBiosciences

CD11b (M1/70) FITC 1:100 Rat IgG2b eBiosciences

Streptavidin PECy7 1:200 eBiosciences

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