Table 4. Time considerations.

Basic protocol 1
Protocol step	Duration	Additional notes
Thaw and passage cells	15 min / passage	Start at least 1 week before surgery; account for extra time when preparing sterile culture medium (30 min).
Thaw Matrigel	Overnight	1 day before surgery.
Prepare and provide non-medicated jelly (optional)	60 min	1 day before surgery; account for cool-down time (10 min) and refrigerator time (45 min).
Count and prepare cells for injection	30 min	Day of surgery, to be done by assistant while surgeon prepares the room.
Room and surgical field preparation	30 min	Day of surgery, to be done by surgeon while assistant prepares cells for injection.
Pre-operative care (mice)	30 min	Day of surgery, to be done by assistant while lead surgeon transports cells to animal facility.
Surgery	10 min / mouse	Account for more time in case of complications, or less time as familiarity with the procedure increases.
Prepare and provide medicated jelly (optional)	60 min	Day of surgery; account for cool-down time (10 min) and refrigerator time (45 min).
Clip removal	10 min	Day 5 post-surgery.

Alternate protocol 1
Protocol step	Duration	Additional notes
Thaw and passage cells	15 min / passage	Start 1-2 weeks before surgery; account for extra time when preparing sterile culture medium (30 min).
Thaw Matrigel	Overnight	1 day before surgery.
Count and prepare cells for injection	30 min	Day of surgery, to be done by lead surgeon while assistant prepares room.
Room preparation	10 min	Day of surgery, to be done by assistant while lead surgeon prepares cells for injection.
Cell injection	5 min / mouse	Account for less time as familiarity with the procedure increases.

Basic protocol 2
Protocol step	Duration	Additional notes
Prepare buffers	30 min	Thaw collagenase II, collagenase IV, DNase, and FBS; bring HBBS to room temperature. Prepare digestion cocktail and R10.
Label and coat tubes	5 min
Euthanize mice and collect blood	3-5 min / mouse	Keep at room temperature until further processing.
Harvest tumor and organs	3-5 min / mouse	Keep on ice until further processing.
Tumor and liver: mechanical dissociation	3-5 min / organ
Tumor: enzymatic digestion	30 min	Bring the thermomixer to 37°C prior to start, incubate alongside liver samples.
Liver: enzymatic digestion	10 min	Bring the thermomixer to 37°C prior to start, incubate alongside tumor samples.
Tumor and liver: straining	20 min	Including sequential elution and a 5-min centrifugation step.
Tumor and liver: density gradient separation	45 min	Including time to slowly overlay cells onto Ficoll (5 min / sample).
Tumor and liver: seed low-density cell fraction in staining plate.	15 min	Including a 5-min centrifugation step and cell count.
Spleen: mechanical dissociation	3-5 min / organ	This and the following steps can be performed during tumor and liver density gradient separation as familiarity with the procedure increases.
Spleen: straining	20 min	Including sequential elution and a 5-min centrifugation step.
Spleen: seed cells in staining plate.	15 min	Including a 5-min centrifugation step and cell count.
Blood: RBC lysis	10 min	Including preparation of 1X working concentration of RBC lysis buffer. This can be performed concomitantly with spleen processing as familiarity with the procedure increases.
Blood: seed cells in staining plate.	15 min	Including a 5-min centrifugation step and cell count

Basic protocol 3
Protocol step	Duration	Additional notes
Prepare reagents	Variable (approx. 30 min)	Mandatory: thaw and dilute LIVE/DEAD Blue and prepare staining buffer. Optional: Prepare Fc block, fixation and permeabilization agent, permeabilization buffer 1X, as well as surface and intracellular antibody master mixes. These reagents can be prepared either prior to the start or during later incubation steps. Store at 4°C, protected from light if appropriate.
Incubate cells with viability dye	40 min	Including washes.
Incubate cells with Fc block	5 min
Incubate cells with surface antibody master mix	40 min	Including washes.
Incubate cells with fixation and permeabilization agent	40 min	Including washes.
Incubate cells with intracellular antibody master mix	40 min	Including washes.
Acquire samples, export .fcs files and analyze	Variable