TITLE: Cell line block and  microarray preparation
AUTHORS: Rona Ortenberg, Erez Nissim Baruch

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol allows to prepare paraffin block of any cell line or co-cultured cells and subsequently these blocks could be used for cell microarray construction. Slides are suitable for ICH/IF staining procedures.</div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Cell microarray can be used, exactly like a tissue microarray, as a high-throughput tool for omics validation or investigation of functional dynamics of targets during a prolonged cell-cell interaction or any other treatments. </div></div></div>

[STEPS]
?. Maintain cell lines in proper medium at 37°C in a humidified atmosphere of 5% CO2 in air.Assess cell confluence. If confluence is 70-80%, proceed.
?. Harvest adherent cells with trypsin solution or cell dissociation solution.Note:Harvesting cell with a cell scraper is not recommended, since it will lead to morphological damage of the cells or the death of a significant portion of the cells. Harvest cells growing in suspension using a mechanical force (pipettor).
?. Count cells.The amount of cells per block should be addusted according the size of the cells.For example , take10-30 million of melanoma  cells or 100-150 million of lymphocytes.
?. Centrifuge cells for 5 minutes at 500 x g, 4°C.
?. Discard supernatant.Re-suspended pellet with  15 ml cold PBS. Centrifuge cells again for 5 minutes at 515 x g, 4°C.
?. Discard supernatant
?. Prepare fixation solution ( in a chemical hood) according to the next steps:
?. Re-suspend pellet with a 15mL of fixation solution and incubate  for 5 minutes in room temperature (chemical hood).
?. Centrifuge 5 minutes at 1250 x g,  4°C.  Discard supernatant.
?. Re-suspend cells with the smallest possible volume of cold PBS and transfer them into one well in a 96-wells U-shaped plate (Cellstar, Sigma-Aldrich).
?. The volume of each of the wells in such plate is ~280 microL. If further PBS volume is needed to transfer the cells into the well, centrifuge the plate for 5 minutes at 515 x g,  4°C and then discard the supernatant, hence making space for additional cells.
?. Once all cells were transferred into the well, centrifuge the plate for 5 minutes at 515 x g,  4°C and then discard the supernatant.
?. Mix the pellet with 100-200 microL of the melted  0.5% agarose gel according to the next steps:
?. Transfer the agar-cell pellet to the appropriate tissue embedding cassette designated for cell block as described below:
?. Embed in paraffin using standard conditions for surgical biopsies.This phase could be done by  the pathological institution and usually takes 48 hours.
?. Cut two 4 micron-thick slices: one for regular H&E stain and one for the specific stain.
?. Stain the H&E slide. The cells should be spread homogenously in the block.
?. Use the Tissue Microarrayer (Beecher Instruments) and a 2-mm-gauge needle.
?. Drill 3 cores from each donor cell block to the recipient cell-line microarray (CMA) block.
?. When the CMA is completed, heat it to 37°C in the designated oven to incorporate the cores into the wax.
?. Stain every 40th CMA section for H&E to make sure that all cores are in place.
?. Take 25mL of 4% PFA
?. Add   24mL of 100% ethanol
?. Add  1 mL of  distillated water
?. Mix well
?. Draw the necessary agar gel volume directly from the melted agar in the warmed bath.
?. Quickly re-suspend cell pellet using the agar and transfer the new suspension into a new flat-bottom 96-wells plate. The flat-bottom is needed creating a unified cell block structure.
?. Cool the new 96-wells plate in 4°C for 45 minutes.
?. Use a sterile scalpel (preferably a straight tip scalpel, as in scalpel #15).Gently insert the scalpel to the small space between the solid agarose-cells structure and the well's wall. Apply a very gentle force sideways. The entire solid mix should pop-out of the well. Note: if you did not stir properly while injecting the melted agar, a portion of the cell will be stuck at the bottom of the well and will be lost. A properly stir mix will leave no trace at the well.
?. Put the solid cells-agarose structure in a cassette, designated for cell blocks. The cassette can be for several hours in a tank full of 4% formaldehyde before final wourkup.