TITLE: Single-Cell Dissociation of Human Trabecular Meshwork 
AUTHORS: Alexi Mcadams, Tavé van Zyl

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Fresh trabecular meshwork tissue is dissected from post-mortem human eyes and dissociated into a single cell suspension.</div></div>

[STEPS]
?. [Prepare Working Solutions and Materials]
Prepare Fresh Oxygenated Ames' MediumAdd 1.9 g sodium bicarbonate to a 1L container (e.g. glass bottle or flask)Empty one container of Ames' Medium powder to 1L containerFill container with 1L of ddH2O, shake to mixBubble oxygen gas (95% O2/5% CO2) through solution
?. [Prepare Working Solutions and Materials]
Prepare papain solutionOxygenated Ames'                                                       10 mLPapain                                                                      200 Units      (ex: for a 44.8 mgP/mL, 26.6 u/mgP batch, use 168 uL)DNase I stock solution                                               100 uLFilter into new 15 mL falcon tube with 0.22 um filter to sterilizeL-cysteine aliquot (see above, stock solution)       100 uLFilter into new 15 mL falcon tube with 0.22 um filter to sterilizeActivate the papain by adding the L-cysteine and incubating at 37 ˚C for 15 minutes before using for dissociation.
?. [Prepare Working Solutions and Materials]
Prepare 1X Ovomucoid solution (LO)10X LO   (aliquot)                                                       1 mLOxygenated Ames'                                                     9 mL
?. [Tissue collection & dissection]
Set up dissection areaClean all intruments and workspace with RNaseZap then 70% EtOH. Place all instruments on a Chux Pad or similar sheet to work on Note: Keep the eye in immersed in Ames media or Optisol within a container on ice until dissection; and preferrably, the eye should be processed within 6 hours from death.
?. [Tissue collection & dissection]
Start Dissection under microscope: Isolate the anterior segmentMake a small incision at the pars plana (~4 mm posterior to the limbus) with a razor blade.Cut circumferentially with curved scissors to liberate the anterior portion from the rest of the eye.  Place the anterior segment in oxygenated Ames' medium.
?. [Tissue collection & dissection]
Expose the trabecular meshworkFree the lens from the ciliary body by carefully cutting the zonules with curved microscissors all the way around the lensGently lift the lens from the rest of the structures with the scissor blades (holding the scissors parallel with the workbench)Take care and try to avoid violating or puncturing the lens capsuleGently peel the ciliary body and iris from the corneoscleral button with forceps.Trim any large chunks of ciliary muscle left behind from the trabecular meshwork with microscissors.This is an ideal time to begin activating the papain solution in a 37˚C waterbath
?. [Tissue collection & dissection]
Isolate the trabecular meshwork Identify the TM and insert one prong of the Jewelers forceps into Schlemm’s canalGrab the TM with the forceps and gently pull the TM out in strips. Some of the ciliary muscle and possibly descemet membrane will come along, and this is okay for the purposes of this protocol The back wall of schlemm canal can also be gently scraped so that cells here can be transferred to the papain solution
?. [Dissociate tissue into single cell solution]
Digest tissueAdd TM strips to prewarmed, activated papain solution in an eppendorf tubeIncubate for a total of 30 minutes at 37 ˚CEvery 7-10 minutes, invert tube to agitate gently
?. [Dissociate tissue into single cell solution]
Dissociate tissue into a single cell suspensionWhen finished incubating, centrifuge digested tissue and papain solution for 30 seconds at 500 rcfCarefully remove supernatant to leave behind tissue Add 1 mL 1X LO to the tissue Triturate with a 1000 uL pipette Filter through a 40 um strainer into a 50 mL falcon tubeIf any large chunks of tissue are left behind, return to eppendorf and add another 1 mL of 1X LO and repeat.Rinse filter with 500 uL - 1000 uL of 1X LO and collect in the same 50 mL falcon tube
?. [Dissociate tissue into single cell solution]
Prepare single cell solution for loading onto 10XPlace the 50ml falcon tube containing the cell/1X LO suspension in centrifugeCentrifuge cells suspension at 500 rcf for 5 minutes (preferably at 4 ˚C) Carefully remove supernatant. Note where pellet should be given the falcon tubes' position in the centrifuge; sometimes the pellet is too small to seeResuspend the cells in 50 uL - 200 uL 0.04% non-acetylated BSA, depending on pellet sizeTransfer to a 1.7 mL eppendorf tubeCount cells with hemocytometer and dilute with 0.04% non-acetylated BSA to desired concentration based on 10X protocol (we usually aim to achieve a concentration of 1000 cells per microliter and load the 10X for 6000 cells recovered)Cell viability assay can be performed at this stage as well prior to loading
?. [Prepare Stock Solutions]
DNase stock solutionDNase I                                                                 40,000 Unitsoxygenated Ames' solution                                             3 mLFilter solution into new 15 mL falcon tube with 0.22 um filter to sterilizeStore 115 µL aliquots at -20˚C.Filter solution into new 15 mL falcon tube with 0.22 um filter to sterilize
?. [Prepare Stock Solutions]
L-cysteine stock solutionL-cysteine, anhydrous (sigma C1276):                    24 mgddH2O                                                                             1 mLFilter into new 15 mL falcon tube with 0.22 um filter to sterilizeStore 115 µL aliquots at -20˚C.Filter into new 15 mL falcon tube with 0.22 um filter to sterilize
?. [Prepare Stock Solutions]
0.4% non-acetylated BSA stock solutionNA BSA                                                                        25 mgoxygenated Ames' solution                                   6.25 mLFilter solution into new 15 mL falcon tube with 0.22 um filter to sterilizeStore 100 µL aliquots at -20˚C.Filter solution into new 15 mL falcon tube with 0.22 um filter to sterilize
?. [Prepare Stock Solutions]
Prepare Oxygenated Ames' MediumAdd 1.9 g sodium bicarbonate to a 1L container (e.g. glass bottle or flask)Empty one container of Ames' Medium powder to 1L bottleFill bottle with 1L of ddH2O,  mix wellBubble oxygen gas (95% O2/5% CO2) through solution
?. [Prepare Stock Solutions]
Prepare 10X Ovomucoid solution (LO)BSA                                                                            150 mgOvomucoid                                                               150 mgOxygenated Ames'                                                     10 mLdissolve and adjust pH to ~7.4 with a few µL of NaOH (2N)Filter solution into new falcon tube with 0.22 um filter to sterilizeMake 1 mL aliquots & store at -20˚C.Filter solution into new falcon tube with 0.22 um filter to sterilize
?. [Prepare Working Solutions and Materials]
Prepare 0.04% non-acetylated BSAOxygenated Ames'                                                     900 uL0.4% non-acetylated BSA stock                               100 uL