This video describes the non-enzymatic dissociation of fresh human tissue to obtain viable cells. The technique is used for qualitative and quantitative analysis of CD45 positive cells (lymphocytes/leukocytes) present in various normal and malignant human tissues.
Protocol
1. Preparation of the Tissue Homogenate Dissect resected tissues (malignant and normal tissue resected from the operating room) are in the pathology lab by trained personnel for immediate pickup. Tumor, NANT (taken the furthest distance from the tumor as possible) and normal tissue fragments are routinely processed within 1 – 3 hr of surgical excision in a BSL2 laboratory using standard biosafety procedures for human tissues. A flow chart of the protocol is illustrated in Figure 1. …
Representative Results
Figure 1: Protocol flow chart. Our procedure for processing fresh human tissues and some analytical approaches that can subsequently be used to assess lymphocytes infiltrating human tissues are shown. Figure 2: H&E stained image of a breast tumor tissue imprint. This impr…
Divulgations
The authors have nothing to disclose.
Materials
GentleMacs Dissociator
Miltenyi Biotec
130-093-235
BD Medimachine is somewhat equivalent
Centrifuge 5810 R
Eppendorf
or other standard table top centrifuge
Centrifuge 5417 R
Eppendorf
or other standard microcentrifuge
Esco Class II A2 Biosafety Cabinet
ESCO global
or other standard BSL2 hood
Inverted Microscope
Nikon eclipse TS100
or other microscope compatible for a hemacytometer